text:
# Design and Folding of Dimeric Proteins

## I Introduction

A large number of proteins perform their biological activity under the shape of dimers (or oligomers). A dimer is a protein whose native conformation is a globule build out of two disjoint chains. Depending whether the two chains have the same sequence or not, they are referred to as homodimers or heterodimers. Notwithstdanding this difference, they have been observed to fold through two major paradigms***A third paradigm, that of ”domain–swapping”, has been described in detail by Eisenberg and coworkers . In the present work we concentrate only on the other two kinds of dimerization. . Some of the known dimers fold according to a three–state mechanism (D$``$I$``$N), where first the denaturated chains of the monomers (D) assume conformations rich of native structures independently of each other (I: folding intermediate), and subsequently the two parts come together to form the dimer (N: native). This is the case, for example, of aspartate aminotransfease , where one can observe three populated species, namely unfolded monomers (D), partially folded monomers (I) and folded dimers (N). A different behaviour is displayed by, for example, P22 arc repressor , whose chains dimerize without populating any monomeric native–like intermediate (two–state process, D$``$N). In this case one can only identify the unfolded monomers (D) and the native dimers (N).

The aim of the present work is to achieve, using model calculations, a basic understanding of the folding mechanism of dimers based solely on energetic arguments, as was already done in the case of small, single domain proteins (monomers). In this case, it was found that good folders are those sequences whose total energy in the ground state conformation lies below a threshold value $`E_c`$ . This threshold energy is solely determined by the number of amino acids forming the chain, the standard deviation of the contact energies used in designing the sequences and by their composition. It is equal to the lowest energy that random heteropolymers of the same length and composition can achieve when they compact into the ensemble of conformations structurally dissimilarThat is, the structures having a small value ($`0.4`$) of the similarity parameter (order parameter) $`q`$, defined as the ratio between the number of native contacts present in the structure to which the sequence has compacted, and the total number of contacts of the native structure used to design the sequence. In the case of dimers, it is convenient to introduce two similarity (order) parameters for any given conformation, namely $`q_1`$ and $`q_t`$ which correspond to the relative value of native contacts within each chain and across the two chains, respectively. to the native conformation.

Aside from being responsible for the thermodynamical uniqueness and stability of the ground state, the low–energy character of single domain good folders is also essential for their dynamics. The only way a small, single domain lattice designed protein can display an energy below $`E_c`$ is by positioning (few) strongly interacting amino acids in some key sites of the protein . These sites, called ”hot” sites in ref.  participate in the formation of local elementary structures (LES), which bias the chain to its native conformation  and which build the (post–critical) folding nucleus when they get together, responsible not only for the stability of the protein, but also of its fast folding ability. Amino acids in these sites are highly conserved in evolution  and determine the topology of the space of folding sequences .

In the following we will characterize the dynamic behaviour of model homodimers with respect to the energetic properties of their ground state conformation. Since a reliable potential function for residue–residue interaction is not available, we can compare the results of model calculations to real proteins only through the analysis of conservation patterns in families of analogousThat is, sequences folding to the same dimeric conformation but not connected by evolution. Couples of sequences whose similarity is lower than $`20\%`$, measured as percentage of matching residues, are likely to be analogous . dimers. Such an analysis has been performed for the analogs of aspartate aminotransfease and of P22 arc repressor, representative examples of a three–state and a two–state folding homodimer, respectively. The corresponding results are displayed in Figs. 1 and 2, and discussed in Section V, where they are compared with the conservation patterns found in lattice model proteins.

## II Design of Sequences

The model we use to study homodimers has been largely employed with success  in the study of monomers. This is because, in spite of the strong simplifications introduced in the description of the proteins, aimed at making feasible dynamical simulations of the folding process, the model still contains the two main ingredients which are at the basis of the distinctive properties of proteins: polymeric structure and disordered interactions among the amino acids. The model is used to study the general thermodynamical and kinetical properties of notional dimers, independently on details concerning secondary structures, side chains, etc. Of course, all these details may prove of relevance when addressing specific questions, as was e.g. done in refs.  concerning the oligomerization equilibrium properties of leucine zippers.

According to the model, a protein is a chain of beads on a cubic lattice, each bead representing an amino acid (selected from twenty different types) which interacts with its nearest neighbours through a contact potential with numbers taken from the statistical analisys of real proteins carried out by Miyazawa and Jernigan in ref.  (for details about the model see, e.g., refs. ).

In the case of single domain proteins it is, in principle, simple to design sequences which fold to a given target conformation. Due to the fact that the thermodynamical and kinetical behaviour of a protein are essentially determined by its total energy, it is possible to design folding sequences by searching in the space of sequences for those having energy lower than $`E_c`$. Using a Monte Carlo algorithm, a sequence with energy $`E`$ in the target conformation has a probability to be selected proportional to $`\mathrm{exp}(E/\tau )`$, where $`\tau `$ is an intensive variable which plays the role of temperature and gives the degree of bias towards low–energy sequences. In the evolutionary context, $`\tau `$ has the meaning of selective pressure with respect to the protein ability to fold : the lower is the value of $`\tau `$, the stronger is this pressure and the better are the folding properties of the selected sequences. In particular, for values of $`\tau `$ lower than the temperature $`\tau ^c(S/E|_{E=E_c})^1`$ (which is the temperature at which the mean energy is exactly $`E_c`$), the average energy of the selected sequences is lower than $`E_c`$. Consequently one obtains in this way sequences with a unique and stable native conformation and able to find it rapidly. In other words, the designed sequences display a unique ground state (native conformation) with energy $`E_N<E_c`$ into which they fold fast .

In order to design homodimers, we first set a target conformation built out of two identical parts (chosen equal to the native structure of a 36mer widely used in the literature in studies of small, single domain proteins ), having a face in contact (see Fig. 3). In the present model the monomers have been chosen to be mirror images of each other, consequently the overall structure is symmetrical with respect to the interface, a feature which simplifies the computational handling of the protein§§§ Strictly speaking, real homodimers are composed of two identical (not symmetrical) monomers. On the other hand, from an energetic point of view our choice is of no consequence. This is because the energy content of a model conformation is solely characterized by its interaction map. Concerning the entropy of the dimer, our choice of native conformation implies a (two–fold) degenerate ground state. Because, this doubling of the degeneracy affects equally all the states of the system, it is of no consequence for the results reported below..

The second step in the homodimer design is to choose a realistic ratio between the different kinds of amino acids and two evolutionary temperatures, $`\tau _1`$ and $`\tau _t`$, which control the amino acids in the bulk and at the interface, respectively. We then use a multicanonical sampling algorithm (see Appendix A) to select a set of sequences $`\{s\}`$, according to the distribution of probability

$$p(\{s\})=\frac{1}{Z}\mathrm{exp}\left(\frac{H_1(\{s\})}{\tau _1}\frac{H_t(\{s\})}{\tau _t}\right),$$
(1)
where $`H_1(\{s\})`$ and $`H_t(\{s\})`$ are the energies associated with the contacts between residues belonging to the same monomer and between residues across the interface, respectively, and $`Z=_{\{s\}}\mathrm{exp}(H_1/\tau _1H_t/\tau _t)`$ is the partition function. During the sampling process, couples of residues belonging to one of the two monomers are swapped, the same swap being repeated on the other monomer, in such a way that the sequence remains identical in the two parts of the dimer. Moreover, in this way the overall concentration of the different kinds of residues is kept constant.

Starting from a random sequence displaying a realistic composition (i.e. the average ratio of different kinds of amino acids as found in natural proteins ), we make $`10^6`$ swappings, sufficient to ensure a stationary distribution of energy, before selecting a sequence, repeating this process $`10^4`$ times, to obtain a statistically representative set of designed sequences $`\{s\}`$. Consequently, in constructing this set we sample only $`10^{10}`$ sequences of the total of $`20^{36}=10^{46}`$ sequences. In keeping with the fact that each of the $`10^4`$ selected sequences are separated from each other by $`10^6`$ swappings, it is reasonable to assume that $`\{s\}`$ is a set of evolutionary uncorrelated sequences whose properties (e.g., conservation patterns) can be compared to those of non–homologous sequences (analogous) families of real proteins.

The use of two different temperatures $`\tau _1`$ and $`\tau _t`$ to select residues in the bulk and at the interface respectively, corresponding to two different evolutive pressures, allows one to control how the energy, and the amino acid conservation pattern, is distributed inside the dimer. Details and caveats concerning the details and limitations of the design procedure are given in Appendix B. Examples of sequences selected at different evolutionary temperatures to fold to the structure displayed in Fig. 3 are listed in Table I.

## III Equilibrium Properties of Sequences

In the present and in the next Sections we shall study the properties and the behaviour of the designed sequences in conformation space, making use of long Monte Carlo runs. In what follows we shall (mainly) concentrate on the thermodynamical properties of these sequences, while in Sect. IV we dwell primarily on their dynamical behaviour.

We first proceed to the calculation of the ground state energy of the designed sequences. Because a complete enumeration of all possible homodimer conformations is out of question, the thermodynamical properties of the sequences selected at different values of $`\tau _1`$ and $`\tau _t`$ (see e.g. Table I) are analyzed through a standard Metropolis algorithm  in the conformational space at fixed temperature $`T`$ (temperature in ”real” conformational space, not to be confused with the ”evolutionary” temperatures $`\tau _1`$ and $`\tau _t`$ in the space of sequences). The simulations were performed with periodic boundary conditions, i.e. in a cubic Wigner cell of dimension $`L`$ (cf. App. C). The need for using a finite volume arises from the fact that, in an infinite volume each conformation with disjoint chains have an infinitely negative free energy, due to its infinite translational entropyIn fact, the entropy of the disjoint system is proportional to $`\mathrm{ln}\mathrm{\Omega }^2`$, while that of the dimer is proportional to $`\mathrm{ln}\mathrm{\Omega }`$, $`\mathrm{\Omega }`$ being the volume allowed to the protein to move in..

Long Monte Carlo simulations ($`510^8`$ MC steps) were performed for each pair of (identical) designed chains. Two outcomes were observed: (A) folding into the dimeric native state, (B) aggregation into a set of disordered clumps. In what follows we shall only analyze the outcome (A), while outcome (B) will be discussed in a forthcoming paper . In Fig. 4 we show the results obtained by carrying out the MC simulation at $`T=0.24`$, for which the fractional population of the native state is $`55\%`$. Full simbols correspond to sequences displaying the behaviour (A), while empty symbols correspond to sequences displaying the behaviour (B). The solid line indicates the loci where the values of $`\tau _1`$ and $`\tau _t`$ correspond to the energy $`E_c=35`$. We note that this line delimits the area in which the corresponding designed sequences display the behaviour of type (A). This is not surprising, considering the fact that the lowest energy of a random conformation with the same number $`n=92`$ of contacts as the target conformation is $`E_c=n\sigma (2\mathrm{ln}\gamma )^{1/2}=34.7`$ (evaluated in the approximation of the random energy model , $`\sigma =0.3`$ being the standard deviation of the interaction matrix elements and $`\gamma =2.2`$ the effective coordination number ). As in the case of single domain proteins, conformations with energy lower than $`E_c`$ have not to compete with the sea of random structures, and consequently are good native conformations (unique and stable)From the experience accumulated on lattice designed, small single–domain proteins, it was learned that the condition that good folders correspond to sequences displaying a sufficiently low energy $`E_{design}`$ in the native conformation (i.e. $`\xi =(E_{design}E_c)/\sigma 1`$, quantity closely connected with the z–score , and where $`\sigma `$ is the standard deviation of the interaction matrix) is actually more general . In fact, because these monomers fold through local elementary structures (LES) stabilized by few (hot), strongly interacting amino acids, and because these amino acids are conserved for all sequences with $`E_{design}<E_c`$, one can restate the large energy gap paradigm of good folders as follows: sequences which display a very small number of hot amino acids and which conserve, in any way, the energy gap . .

In what follows we shall discuss, making again use of the results of long Monte Carlo runs\**\**\**The runs were carried out in a cubic Wigner cell of sidelength $`L=7`$ and periodic boundary conditions because this was found the most appropriate from numerical considerations (cf. App. C)., the properties of two of the sequences shown in Table I, namely sequences $`\mathrm{\#}1`$ and $`\mathrm{\#}3`$, chosen as representative examples of chains building homodimers which fold according to a two– and to a three-state mechanism, respectively. They were both designed at low $`\tau _t`$ and $`\tau _1`$, in the first case with $`\tau _t<\tau _1`$ and in the second case with $`\tau _t>\tau _1`$. In both cases, the designed sequences display a first–order transition in conformational space from the denaturated state into the native dimeric structure, as in the case of single domain proteins, as testified by the discontinuous behaviour of the similarity (order) parameters at the critical temperature $`T^c`$. In other words, the order parameters $`q`$ display at $`T=T^c`$ a double peak behaviour.

As a rule, the critical temperatures $`(T^c)_1`$ and $`(T^c)_t`$ in conformational space will be different. In particular, in the case of sequence $`\mathrm{\#}1`$ of Table I, $`(T^c)_1<(T^c)_t`$, in keeping with the fact that this sequence was designed setting $`\tau _1`$ larger than $`\tau _t`$. This property has the consequence that the native state is stable at temperatures $`T<(T^c)_10.24`$, $`(T^c)_1`$ being the critical temperature, at which the area of the peak associated to the unfolded state ($`q_1`$ small) is equal to that associated to the native state ($`q_1=1`$), and that dimerization happens already at $`T<(T^c)_t0.26`$. The fact that $`(T^c)_1<(T^c)_t`$ indicates that while it is possible to find a situation (at $`T`$ such that $`(T^c)_1T<(T^c)_t`$) in which the two chains build the native interface but the monomers are not folded (cf. e.g. Fig. 5(b) ), there is not a temperature at which the monomers are folded and separated (cf. Fig. 5(a) ).

Also sequence $`\mathrm{\#}3`$ undergoes a first order transition, but in this case the critical temperature $`(T^c)_1`$ associated with the bulk is larger than that associated with the interface ($`(T^c)_1=0.25>(T^c)_t=0.22`$). This indicates that for sequences selected at comparatively high selective temperature $`\tau _t`$ ($`=10\tau _1`$, cf. Table I), there exists a phase where the two chains are folded but separated (cf. Fig. 6).

Another interesting thermodynamical property of a dimer is the localization of the sites which are mostly responsible for the stabilization of the native state. These sites can be identified by the change in energy $`\mathrm{\Delta }E_{loc}`$ that mutations induce in the native conformation . For a given site $`i`$, 19 mutations can be carried out. Because $`\mathrm{\Delta }E_{loc}(i)`$ may change markedly depending on the point mutation introduced in the protein, it is useful to define the average value $`\overline{\mathrm{\Delta }E_{loc}(i)}`$ taken over all possible substitutions. In keeping with the result of studies of single domain protein , in particular of the sequence $`\mathrm{\#}6`$ of Table I, ”hot” sites (sites which are most sensitive to point mutations, which as a rule, if mutated, denaturate the protein) are those sites for which $`\overline{\mathrm{\Delta }E_{loc}(i)}>\mu +2\sigma `$, where $`\mu `$ is the average value of $`\overline{\mathrm{\Delta }E_{loc}(i)}`$ ($`i=1,\mathrm{},N`$) taken over all the sites of the chain, while $`\sigma `$ is the associated standard deviation. ”Cold” and ”warm” sites in the nomenclature of ref.  are those sites for which $`0\overline{\mathrm{\Delta }E_{loc}(i)}\mu `$ and $`\mu \overline{\mathrm{\Delta }E_{loc}(i)}\mu +2\sigma `$ respectively††††††Note that this general definition essentially coincides with that given in ref.  for the case of S36, i.e. $`\overline{\mathrm{\Delta }E_{loc}(i)}<1`$ (cold), $`1\overline{\mathrm{\Delta }E_{loc}(i)}<2`$ (warm), $`1\overline{\mathrm{\Delta }E_{loc}(i)}>2`$ (hot), in keeping with the fact that, in this case, $`\mu 1`$ and $`\sigma 0.7`$..

In Fig. 7 we display the values of $`\overline{\mathrm{\Delta }E_{loc}(i)}`$ associated with sequence $`\mathrm{\#}1`$, $`\mathrm{\#}3`$, $`\mathrm{\#}5`$ and $`\mathrm{\#}6`$ (S36 monomer) of Table I. A marked difference in the pattern distribution of hot and warm sites associated with sequence $`\mathrm{\#}1`$ (two–state folding) and $`\mathrm{\#}3`$ (three-state folding) is observed. In fact, sequence $`\mathrm{\#}1`$ (Fig. 7(a) ) displays no hot sites and double as many warm sites than sequence $`\mathrm{\#}3`$ (Fig. 7(b) ), the properties of the sites of this last chain in the native conformation being essentially those found in the study of the isolated monomer S36 (Fig. 7(d), cf. also ). Furthermore, the amino acids occupying the warm sites of chain $`\mathrm{\#}1`$ are, in average, more strongly interacting (2.4) than those associated with the warm sites of chain $`\mathrm{\#}3`$ (1.9), althoug still much less than hot sites of this chain (3.1). In other words, in sequence $`\mathrm{\#}3`$ most of the binding energy is concentrated in few ”hot” sites, while in the case of sequence $`\mathrm{\#}1`$ the stabilization energy is spread more homogeneously throughout the monomers.

## IV Dynamics of dimerization

We now concentrate our attention, making again use of the results of the Monte Carlo simulations already discussed in the last Section, on the dynamics of the process that leads the system from a random conformation to the native dimer (for details and caveats see Appendix B). The first issue to assess is whether the dimeric native state is accessible on a short time scale (short with respect to the random search time, $`10^{50}`$ MC steps). For each of the sequences selected at various evolutionary temperatures (some of them being listed in Table I), 20 simulations of $`6010^6`$ MC steps have been performed, recording the first passage time, i.e. the folding time $`t_f`$ (cf. Table II) into the dimeric native conformation. Each set of simulations has been repeated at three temperatures, $`T=0.24`$, $`T=0.28`$ and $`T=0.32`$.

We shall first discuss the case of sequences optimized at very low values of both $`\tau _1`$ and $`\tau _t`$ and with $`\tau _t<\tau _1`$, so that the interface energy is close to its global minimum (solid squares in Fig. 4). This design procedure was followed in the expectation to obtain sequences which fold through a two–state process, namely first dimerization and then folding. To check this scenario, we have determined through dynamical MC simulations the distribution of $`q_1`$ (and $`q_t`$) associated with sequence of type $`\mathrm{\#}1`$ (cf. Table I), and calculated at the instant in which $`q_t`$ (or $`q_1`$) reach, for the first time, a value of the order of one‡‡‡‡‡‡For computational easiness, and because the parameters $`q_1`$ and $`q_t`$ vary discontinuously as a function of the number of MC steps, we have used the MC step at which a value of $`q_1`$ or $`q_t`$ larger than $`0.7`$ is reached for the first time to define the instant at which to calculate the value of the other order parameter (and thus the associated distribution) $`P(q_t)`$ or $`P(q_1)`$ respectively.. The results displayed in Fig. 8(a) show that, by the time the bulk of the two chains fold, $`q_t`$ is essentially equal to 1, indicating that the interface between the two chains is already in place. On the other hand, at the time in which $`q_t`$ acquires for the first time a value $`1`$, $`q_1`$ displays a rather flat distribution, its average value being $`0.4`$. One can conclude that sequence $`\mathrm{\#}1`$ of Table I first dimerizes and then folds.

The fact that in the present case the folding time $`\tau _f`$ increases with temperature (cf. Table II) indicates that the dimerization process is not diffusion limited. That is, $`\tau _f`$ is determined not only by the time needed by the two interfaces to come in contact, but also by the stability of the interface structure. In first approximation, the diffusion time $`\tau _D`$ for the interfaces to meet, in the approximation that two interfaces search for each other randomly in the space of configurations, is $`\tau _D(64V)=8200`$ steps, where 6 is the number of faces of an ideal cubic conformation, 4 takes into account the rotational symmetries of the faces and $`V=7^3`$ is the volume of the system. This time is much shorter than the folding time. On the other hand, at $`T=0.28`$ the relative population of an isolated fragment containing the 12 interface residues in place (forming the surface of one of the two monomers) has been calculated to be $`p_{int}=0.018`$ (determined through a MC simulation), so that the probability to have the two interfaces structured at the same time is $`p_{int}^2=3.2410^4`$. Consequently, in this simple model, the folding time is predicted to be $`t_f=t_Dp_{int}^22.510^7`$, which agrees well with the value found in detailed MC simulations and displayed in Table II. This picture explains also the increase of aggregation probability with temperature. In fact, the higher is the temperature, the longer is the time that partially structured surfaces which eventually dimerize move around in configurational space and can bind to wrong partners, causing aggregation.

Also the behaviour of unfolding times agrees with the picture of folding–after–dimerization. In fact, starting from the native conformation, the average decay of $`q_t`$ with respect to time is (cf. Fig. 9) $`t_t=5.8310^5`$ at $`T=0.28`$ (the values for other temperatures are listed in Table II). On the contrary, the distribution of $`q_1`$ is best fitted by a stretched exponential in the form $`q_1(t)=\mathrm{exp}(t^\beta /a)`$, with $`\beta =0.52`$ and $`a=1470`$, the average time being $`t_1=7.9910^5`$. The fact that $`q_1(t)`$ does not follow an exponential law indicates that the unfolding of each of the two chains is not a process which depends only on its internal (intra monomer) contacts, like e.g. in the case of single domain proteins, cf. , but is subordinated to an external event. In keeping with this fact, and because the detaching time $`t_t`$ (the characteristic decay time of $`q_t`$) is shorter than $`t_1`$, one can conclude that the breaking of the native bonds associated with the internal structure of the two chains is a consequence of their detaching or, in other words, that the stability of the monomer structures relies on the presence of the interface.

We now turn our attention to the sequences selected at $`\tau _1<\tau _t`$ in order to be close to the global minimum of the bulk energy ($`E<16`$) and corresponding to the solid triangles in Fig. 4. Sequence $`\mathrm{\#}3`$ of Table I is an example of this class of designed homodimers. The dynamical behaviour of this sequence is quite different from that of sequence $`\mathrm{\#}1`$, although at $`T=0.28`$ it folds with probability $`0.4`$ to its native conformation, in an average time (first passage time (FPT) ) of $`t_f=3.210^7`$ (cf. Table III) MC steps, quantities which is quite similar to those associated with sequence $`\mathrm{\#}1`$ ($`p_N=0.45`$ and $`t_f=2.410^7`$ MC steps, respectively). The analysis of the time dependence of $`q_1(t)`$ and $`q_t(t)`$ shows (cf. Fig. 8(b) ) that for sequence $`\mathrm{\#}3`$ $`q_t`$ is very small at the time in which $`q_10.7`$, while at the time at which $`q_t0.7`$, $`q_1`$ is already essentially equal to 1, indicating a three–state scenario and the esistence of an intermediate state where the two monomers are folded and not dimerized. That is, sequence $`\mathrm{\#}3`$ first fold to its monomeric native conformation and then forms the dimer.

In the folding process, this sequence follows the hierarchical path typical of monomeric model proteins , building first local elementary structures (LES), then the (postcritical) folding nucleus, finding the monomeric native conformation shortly after. In addition, in the present case, there is a further step which consists in the association of the two monomers to build the dimer (to be noted that sequence $`\mathrm{\#}3`$ has the same local elementary structures and folding core than the monomeric protein studied in Ref. , as testified by the fact that the hot sites are essentially the same (cf. Figs. 7(b) and (d) )).

The same sequence, when studied as an isolated chain at the same temperature, folds in an average time of $`0.910^6`$ steps, indicating that the time limiting step is the association (it takes $`2.410^7`$ steps). As temperature increases, the folding time decreases slightly (see Table III). This decrease is much less pronounced than the decrease in the diffusion time as measured by the inverse of the diffusion coefficient $`D^1`$ (which is exponential with temperature, see third column in Table III), indicating that the dimerization time is not purely diffusion limited. On the contrary, the stability of the interface (which depends exponentially on temperature as well) plays an important role in the dimerization time.

To check the generality of the results discussed above, we have repeated the design and the folding for another three dimensional conformation, where the native structure of each of the monomers is the same, but the interface involves the opposite face than in the case of the dimeric conformation shown in Fig. 3. While the folding time of sequences which first fold and then aggregate is similar to that of sequence $`\mathrm{\#}3`$, the folding time for sequences behaving according to a two–state mechanism is substantially higher (of the order of $`10^8`$ MC steps). This is due to the much lower stability of the interface built out of the two, rather unspecific surfaces stabilized by the contacts between residues lying rather distant from each other along the chain (e.g. 19–32, 17–32), while in the previous case the surfaces were pieces of incipient $`\beta `$–sheets stabilized by quartets of residues (e.g. 3–6, 7–10). Nonetheless, also in this case it is possible to find sequences which fold according to the two paradigms discussed above.

Summing up, it is found that if the folding of the homodimer is controlled by stable LES (cf. ref. ) stabilized by few hot, strongly interacting amino acids, the folding time decreases with temperatures, as the folding of the protein is (post–critical) folding–nucleus–formation controlled. In fact, the folding nucleus is the result of the docking of the LES in the appropriate way, and thus controlled by the diffusion time of the LES, a process which is speed up by increasing the temperature. This happens until one reaches a temperature at which the stability of LES is affected, beyond which the folding time rises again. Since the stabilization energy of LES is much larger than the interaction energy between pairs of amino acids , there exists a large window of temperatures at which folding time is short. This scenario reflects, to a large extent, the temperature dependence of the folding time of monomers of which the homodimer is made of (cf. Fig. 10).

The situation is quite different in the case of a dimer built out of two sequences of type $`\mathrm{\#}1`$, which first dimerize and then fold (two–state process). In this case, the folding of the system is not controlled by LES. This is because, were one to put ”hot” amino acids (needed to stabilize the LES) on the surfaces which dimerize, these surfaces will be so reactive that the system will aggregate with high probability (open circles in the low–$`\tau _t`$ region of Fig. 4). But if the dimerizing surfaces do not contain ”hot” amino acids, the folding of the remaining amino acids (”volume”) cannot depend on LES either, otherwise the system will behave more like sequences of type $`\mathrm{\#}3`$. Consequently, if both the dimerizing surfaces and the remaining part of the (”volume”) system are marginally stable, the binding energy being essentially uniformly distributed among all the pairs of nearest neighbours amino acids (low $`\phi `$–values associated essentially with all sites), the fast folding temperature window is expected to be much narrower than in the case of homodimer based on sequence of type $`\mathrm{\#}3`$. In fact, already at low temperature ($`T=0.24`$), the system based on sequences of type $`\mathrm{\#}1`$ is found to be passed the minimum of the curve shown in Fig. 10, and one observes an increase of the folding time as a function of $`T`$ (cf. Table II).

## V Results for real proteins

A possible connection between the model of homodimer folding discussed above and real proteins can be achieved by studying the degree of amino acid conservation in each site of analogous dimers. This can be done by calculating the entropy in the space of sequences in each site, given by

$$S(i)=\underset{\sigma =1}{\overset{20}{}}p_i(\sigma )\mathrm{log}p_i(\sigma ),$$
(2)
where $`p_i(\sigma )`$ is the probability of finding the amino acid of kind $`\sigma `$ at site $`i`$, a probability to be calculated over a large number of sequences folding to the same conformation.

Within the present model, the calculation of $`S(i)`$ is straightforward. Given a dimeric native conformation (Fig. 3), and a couple of evolutionary temperatures $`\tau _1`$ and $`\tau _t`$, one selects from the corresponding set of designed sequences a representative sample (e.g. $`10^4`$) of them. Since these sequences are aligned, it is possible to calculate the associated values of $`p_i(\sigma )`$ and, consequently, $`S(i)`$. The entropy at each site ranges from $`0`$, if only one kind of amino acids occupies that site in all sequences selected, to $`2.996`$, if each kind of amino acid is equiprobable, the latter being the case at large values of the design temperature.

We have shown elsewhere  that, for monomeric proteins, low–entropy sites are those where most of the stabilization energy is concentrated. The search of low–entropy sites rather than of strongly–interacting sites has the advantage that it can be easily performed for real proteins, for which a reliable energy function is not available.

In Figs. 11 and 12 we display the entropy per site and the associated distribution of entropy for: (a) sequences displaying the folding behaviour of sequence $`\mathrm{\#}1`$ (solid squares in Fig. 4), (b) for sequences displaying the folding behaviour of sequence $`\mathrm{\#}3`$ (solid triangles in Fig. 4), (c) aggregating sequences (empty circles in Fig. 4), (d) for the monomer S36, single domain protein, which folds into the native conformation corresponding to half of the homodimer shown in Fig. 3 (sequence $`\mathrm{\#}6`$ of Table I).

One observes a marked difference between the entropy per site shown in Fig. 11(a) and that displayed in Fig. 11(b). In fact, sequences of type $`\mathrm{\#}1`$ have many more conserved sites than sequences of type $`\mathrm{\#}3`$ (the ratio between these two numbers being 3.3), the average entropy being a factor of 1.6 larger than that associated with sequences of type $`\mathrm{\#}3`$. Furthermore, in the case of sequence $`\mathrm{\#}3`$ only $`20\%`$ of the conserved sites lie on the surface, while in the case of sequence $`\mathrm{\#}1`$ this ratio is $`40\%`$. On the other hand, cases (a) and (b) cannot be distinguished by looking at the interface/bulk average entropy, being 1.42 and 1.43 respectively in the case of sequence of type $`\mathrm{\#}1`$ and 1.17 and 1.24 in the case of sequences of type $`\mathrm{\#}3`$. The situation is similar if one looks at the values of the average entropy associated with conserved sites lying on the interface ($`<S_{cons}>_t`$) as compared to the average entropy $`<S_{cons}>`$ associated with all conserved sites (cf. Table IV).

For aggregating sequences (empty circles in Fig. 4, see Fig. 11(c) ), there is an evident difference between the entropy of the interface (sites 1–12), whose average entropy is 1.18, and the bulk, whose average entropy is 1.71. Aggregation arises due to the fact that the surface, which essentially contains all of the ”hot” sites of the protein ($`85\%`$ of them), is too reactive.

Summing up, sequences of type $`\mathrm{\#}1`$ give rise to a dimer where the conservation pattern is distributed much more uniformly among all sites and where there are quite a large number of important sites although much less conserved than the few hot sites of sequences of type $`\mathrm{\#}3`$. These results indicate that, from the conservation patterns of lattice designed sequences, it is possible to recognize sequences which aggregate from those which dimerize. Among these we can recognize sequences which dimerize through a three–state scenario or through a two–state mechanism by looking at the overall distribution of entropy, but not at the difference between the entropy of the interface and of the bulk. These results agree with the findings by Grishin and Phillips, which analyze the conservation of residues on the surface of five oligomeric enzymes and find no signal of any larger conservationism on the interface with respect to the bulk . We suggest that one has to analyze the distribution of entropy, not the bulk/interface partition, to derive the behaviour of a family of sequences.

To test the predictions of the model, we have calculated the entropy at each site for two dimeric proteins, i.e. P22 arc repressor and aspartate aminotransfease (cf. Figs. 1 and 2). The former folds according to the paradigm of sequence $`\mathrm{\#}1`$ of Table I (first dimerize and then fold), while the latter follows the opposite scenario (the monomers first fold and then dimerize). From the fssp database we have selected, for each of the two proteins, a family of non–homologous aligned sequences which have the same fold and the same interface (z–score larger than 2), and from them we have calculated $`S(i)`$. The entropy was calculated in three different ways, in order to take care of the gaps in the alignment. The results indicated in Figs. 1 and 2 in terms of solid lines were obtained considering the positions in the gaps as filled by a kind of amino acid which is, by definition, different from all others (including the other ”fake” amino acids put in the gaps of the other sequences). Dots indicate a calculations where amino acids are grouped into six classes (cf. ref. ), while the dotted line is calculated ignoring sequences which, at a given site, display a gap. Below the x–axis it is displayed, with a black stripe, the interface region.

The relative number of conserved sites is considerably larger (a factor 2.7, cf. Table IV) in the case of P22 arc repressor than in the case of aspartate aminotransfease. Of them, only $`20\%`$ lie on the interface of aspartate aminotransfease while $`37\%`$ does in the case of the two–state P22 arc repressor.

Further confrontation between the results of lattice model calculations and real proteins can be carried out through the entropy distributions associated with Fig. 11 (designed proteins) and Figs. 1 and 2 (P22 arc repressor and aspartate aminotransfease), as displayed in Figs. 12 and 13, respectively. The arc repressor distribution displays an abrupt increase of the entropy at a value somewhat larger than 1 which is the edge of a well defined peak\**\**\**The lowest entropy bin, separated from the others, contain a single site and could be associated with functional purposes. The highest bin, on the other hand, contains all the sites which have gaps in the alignment., a behaviour which is very similar to that of sequences of kind $`\mathrm{\#}1`$ shown in Fig. 12(a). On the other hand, aspartate aminotransfease displays a gradual increase in the distribution of entropy, and a two peak structure, which resembles the corresponding quantity associated with sequences of type $`\mathrm{\#}3`$ (cf. Fig. 12(b) ), having few sites which are highly conserved. These results, as well as those displayed in Table IV, thus seem to confirm the overall picture emerging from lattice model calculations.

We conclude this section by recalling the fact that the data used to derive the numbers displayed in Figs. 1 and 2 (6 and 11 sequences respectively) have rather poor statistics, because not many analogous sequences with the same interface are available in the PDB. Another caveat to be used in comparing the data with the model calculations is the fact that real proteins aside from structural features, display functional properties (not present in model calculations), which can have conditioned the conservation of amino acids at precise sites, in particular surface and interface sites.

## VI Conclusions

In the present paper we have designed, with the help of a lattice model, sequences which either first dimerize and then fold or, conversely, first fold and then dimerize. They were obtained by minimizing their energy in a given (dimeric) conformation with respect to amino acid sequence at constant composition. The swapping of amino acids was carried out making use of Monte Carlo techniques. Two design temperatures have been used to carry out the minimization process, one controlling the evolutionary pressure on the interface amino acids, the other controlling the pressure on the amino acids occupying the bulk of the dimer.

We know that the way evolution solves the protein folding problem of small monoglobular systems, at least within lattice models, is by asigning the commanding role of the process to few, hot, highly conserved amino acids (low entropy ”bump” of Fig. 12(d) ). For the remaining amino acids one can essentially choose anyone among the twenty different types. In fact, we know  that there are $`10^{30}`$ sequences sharing the same ”hot” amino acids which fold to the native structure on which the monomeric sequence S36 (sequence $`\mathrm{\#}6`$, Table I) was designed (high entropy peak of Fig. 12(d)).

The further requirement of dimerization after folding, typical of sequences of type $`\mathrm{\#}3`$, seems to be solved by evolution through an increase in the number of commanding amino acids, that is, by increasing the number of highly conserved, low entropy monomers. In other words, by shifting amino acid (sites) from the high–S peak of Fig. 12(d) into the low–S peak of the same figure, as testified by the entropy distribution associated with sequences of type $`\mathrm{\#}3`$ shown in Fig. 12(b) (cf. also Figs. 7(b) and 7(d) ).

This change in the strategy of evolution is complete in the case of sequences of type $`\mathrm{\#}1`$, which first dimerize and then fold. In this case, all amino acids become essentially equally conserved, the small high–S peak of Fig. 12(b) being absorbed in the long tail of the single peak of Fig. 12(a). This is consistent with the low $`\phi `$–values observed for the transition state of P22 arc repressor .

From these results one can conclude that sequences which first dimerize and then fold must be much more difficult to come about than sequences that first fold and then dimerize, in keeping with the fact that the first type of sequences are dependent on a significantly larger set of conserved amino acids to reach the protein native structure in the folding process, than the second type of proteins.

Appendices

## A On the Selection of Sequences

The unusual feature of the present design process is the fact that it requires different average energies for the bulk and for the interface. As a consequence, it is not possible to apply standard equilibrium thermodynamics. Anyway, one can still proceed in parallel with the canonical ensemble picture and regard the system as composed of two interacting parts, each of them in contact with its own thermal bath. Of course we are not interested in the true equilibrium of the system, which would require the two baths to reach the same temperature and the same average energy, but in a stationary state in which the average energies are constant.

If we call $`p`$ a generic distribution of states of the system, we can define the average energy functional $`E_1[p]=H_1p`$ and $`E_2[p]=H_2p`$ of the two parts of the system and the entropy functional $`S[p]=p\mathrm{log}p`$, which indicates the information we have about the system.

Among all possible distributions $`p`$, we are interested in the stationary distribution $`p^{}`$ which minimizes the information (maximizing $`S`$) at fixed $`E_1[p]=E_1^{}`$ and $`E_2[p]=E_2^{}`$. In keeping also with the constrain $`p=1`$, one finds the distribution

$$p^{}=\frac{1}{Z}\mathrm{exp}(\alpha H_1\beta H_2),$$
(A1)
where $`Z=\mathrm{exp}(\alpha H_1\beta H_2)`$ normalizes the probability to unity and $`\alpha `$ and $`\beta `$ are the Lagrange multipliers which set the average energies. To evaluate them, we can write the entropy $`S[p^{}]`$ as a function of the energies $`S(E_1,E_2)=\alpha E_1+\beta E_2\mathrm{log}Z`$. Its derivatives in $`E_1^{}`$ and $`E_2^{}`$ give

$`{\displaystyle \frac{S}{E_1}}=\alpha ;`$ $`{\displaystyle \frac{S}{E_2}}=\beta .`$ (A2)
In parallel with equilibrium thermodynamics, we call temperatures the inverse of the two Lagrange multipliers, $`\tau _1=\alpha ^1`$ and $`\tau _t=\beta ^1`$.

To select sequences $`\{s\}`$ distributed according to

$$p(\{s\})=\frac{1}{Z}\mathrm{exp}\left(\frac{H_1}{\tau _1}\frac{H_t}{\tau _t}\right),$$
(A3)
where $`H_1`$ and $`H_t`$ are the bulk and the interface energy of the sequence, respectively, and $`Z=_{\{s\}}\mathrm{exp}(H_1/\tau _1H_t/\tau _t)`$, we use a multicanonical technique (see Appendix B).

The multicanonical method  is an extension of the usual Monte Carlo sampling method. In the latter the phase space of a generic system kept at temperature $`T`$ is sampled making trial random moves (here, amino acid swappings) and accepting the move with a probability $`\mathrm{min}(1,\omega (trial)/\omega (old))`$, where $`\omega (state)`$ is the equilibrium statistical weight of a given state of the system, which is of course the Boltzman distribution $`\mathrm{exp}(E(state)/T)`$. This works efficiently at high temperatures, but becomes problematic when $`T`$ is decresed, due to the fact that the system can get trapped in local energy minima. To overcome this problem, the multicanonical method samples the phase space using unconventional statistical weigths, namely $`\omega (state)=1/g(state)`$, where $`g(state)`$ is the energetic degeneracy of that state. Coarse graining the description of the system, each state can be labelled with its energy and the statistical weight associated with a given energy is $`g(E)\omega (E)=1`$. In other words, we are sampling a phase space which (using these artificial weights) is flat, so that the system cannot get trapped in metastable states. The problem is that $`g(E)`$, which defines the weights, is not known a priori. The algorithm has to be, accordingly, self–consistent: a trial $`g(E)`$ is guessed, the phase space is sampled with weights $`\omega (E)=1/g(E)`$, from the results of the sampling a new distribution $`g(E)`$, and so on. When $`g(E)`$ has converged, one has found the distribution of energies of the system (which does not depend on the weights used to find it!) and from this derive all the other thermodynamical quantities of the system.

The algorithm is computationally requiring, also because at each autoconsistent step the information found in the preceding steps, save the last, is discareded. To solve this problem a very efficient method, which combines the informations found during the whole calculation, has be developed by Borg and described extensively in ref. . In the sampling of the space of sequences we have used the multicanonical algorithm and checked the results with the extension of ref. .

## B Simulation of the Dynamics

Strictly speaking, the Monte Carlo algorithm was designed to study equilibrium properties of systems with many degrees of freedom . Nonetheless, it has been shown  that, being equivalent to solving the Fokker–Planck Equation for diffusion in a potential, it can be helpful also in studying the kinetical properties of complex systems, provided that the Fokker–Planck approximation is valid (i.e., moves are local and the potential changes smoothly on the diffusionlenght scale). Furthermore, Rey and Skolnick have shown  that the folding trajectories obtained for single domain proteins with Monte Carlo simulations are consistent with those obtained with real Molecular Dynamics calculations.

In the study of dimers there is an additional problem. Since the Monte Carlo moves are local, it is not evident that this algorithm describes properly the diffusion of one chain with respect to the other. To make sure that the present algorithm is suitable to deal with diffusion, we have simulated the displacement of the center of mass of single monomer heteropolymers. In Fig. 14 it is displayed the mean square displacement of the center of mass $`<x^2>`$ as a function of time for five sequences designed, to different degrees, to fold to the same conformation. It is also displayed the behaviour of a random sequence (the first to the left). The calculations are performed at the temperature $`T=0.28`$ and the average is done over 50 independent runs, each time starting from a random conformation. All these sequences move in a diffusive regime, characterized by $`<x^2>=Dt`$, where $`t`$ is time (measured as number of MC steps) and $`D`$ is the diffusion coefficient.

In the inset we show the diffusion coefficient $`D`$ of each sequence (in units of $`10^4`$ lattice units over number of MC steps) with respect to its native energy (on the horizontal axis is the energy $`E_cE`$). It is clear from this plot that the diffusion coefficient depends on the stability of the protein, a feature which is rather unphysical since it should only depend on the shape of the polymer and on the properties of the solvent. On the other hand, one can notice that for optimized sequences ($`E_cE0`$) $`D`$ decreases linearly with the energy, spanning a range $`2.1110^5MCS^1`$ per unit energy. Consequently, we expect that the effects of this dependence on the folding mechanism of optimized sequences are negligible.

The dependence of the diffusion coefficient on temperature for a fixed sequence (e.g. $`\mathrm{\#}1`$ of Table I) is displayed in Fig. 15. Except for low temperatures ($`T<0.26`$) it satisfies Einstein’s Equation $`D=\mu T`$, where $`\mu `$ is the mobility of the chain, indicating that that chain undergoes Brownian motion.

## C Periodic Boundary Conditions

The choice of the linear dimension $`L`$ of the Wigner cell where the Monte Carlo simulations were carried out was found to be important in determining the dynamical evolution of the system. This quantity is the model parameter which reflects the density $`\rho =2/L^3`$ of sequences in real systems, either the cell or a test tube, which eventually fold in the native conformation of the designed homodimer. If $`L<7`$, we found that the chains get entangled in some non–native conformations, since there is not enough room for them to assume the native conformation (which is a parallelepiped of $`6\times 4\times 3`$). If $`L>8`$ the translational entropy of the disjoint chains is so large that the conformation corresponding to the two chains folded and separated becomes the equilibrium state. In other words, although the two chain system is able to reach the native homodimer conformation, it is very unstable (e.g., the relative population of the homodimer native conformation is, for sequence $`\mathrm{\#}1`$ at $`T=0.28`$ and $`L=7`$, less than $`10^3`$). In what follows we shall set\*†\*†\*†Due to the discrete character of the conformational space, it is not possible to fine–tune the choice of $`L`$ $`L=7`$, even if with this choice, the system experiences some difficulties in reaching the native conformation, due to the narrowness of the space available. For example, in 20 long MC runs ($`510^8`$ MC steps), sequence $`\mathrm{\#}1`$ can find its correct dimeric native state in 16 times out of 20. In fact, in 4 cases it finds a conformation with energy $`E=35.92`$, corresponding to a situation in which one of the two chains (let us call them A and B) is folded (say, chain A), the monomers of chain B at the interface being in their native position, while chain B is in a (well defined) conformation which has only $`40\%`$ similarity with its native structure (similarity parameter $`q=0.4`$). The reason for this result is to be found in the fact that chain B builds some contacts with the ”back” of chain A, taking advantage of the periodic boundary conditions. These contacts are mostly between residues of chain B and partners which are of the right kind but belong to the wrong chain (contacts 17A–32B, 23A–18B, 24A–17B, 25A–36B, 26A–35B, 35A–26B), while two of them are between monomers which cannot be in contact if they belong to the same chain (31A–32B, 32A–33B). While these processes may be of relevance, for example, in connection with the formation of amyloid aggregates, it arises in the present work due to an artifact of the model. In fact, it is connected with the fact that we are simulating a system of many chains by considering explicitely only two of them with periodic boundary conditions. As a consequence, a domain–swapping–like mechanism  is likely to take place. In fact, if after the two chains have built the native interface, a subdomain of one of the two sequences is not in place but moves around, it can find itself in the vicinity of its complement, but belonging to the other chain. We shall come back to phenomena of domain–swapping  in a future publication, where we shall consider more than two chains .


format: text

text:
# Atom–Molecule Coherence in a Bose-Einstein Condensate

## Abstract

Coherent coupling between atoms and molecules in a Bose-Einstein condensate (BEC) has been observed. Oscillations between atomic and molecular states were excited by sudden changes in the magnetic field near a Feshbach resonance and persisted for many periods of the oscillation. The oscillation frequency was measured over a large range of magnetic fields and is in excellent quantitative agreement with the energy difference between the colliding atom threshold energy and the energy of the bound molecular state. This agreement indicates that we have created a quantum superposition of atoms and diatomic molecules, which are chemically different species.

There is considerable interest in extending the applications of ultracold atoms to ultracold molecules. One route for producing a very cold and possibly Bose-condensed sample of molecules is to create the molecules from an atomic BEC. Wynar et al.1 created cold 87Rb2 molecules in a single ro-vibrational state of the ground-state potential from an 87Rb BEC using a two-photon stimulated Raman transition. The authors could not probe the coherence properties of the molecules in that state, but the prospect of creating a superposition of atomic and molecular condensates initiated a flood of theoretical work on the subject2-6. Ultracold molecules have also recently been formed through photoassociation of a sodium BEC7.

Utilizing the natural atom–molecule coupling that arises from a Feshbach resonance is an alternate route for producing ultracold molecules from an atomic BEC, and it is the route we have followed here. A Feshbach resonance is a scattering resonance for which the total energy of two colliding atoms is equal to the energy of a bound molecular state, and atom–molecule transitions can occur during a collision8-12. A schematic representation of the potentials involved is shown in the inset of Fig. 1A. For our 85Rb resonance, BEC atoms in the $`F=2`$, $`m_F=2`$ state collide on the open-channel threshold. $`F`$ and $`m_F`$ are the total spin and spin-projection quantum numbers. The bound state in the closed channel differs in energy by an amount $`ϵ`$ from the open-channel threshold. The bound molecular wave function can be described as a sum of amplitudes of different hyperfine components ($`F`$, $`m_F`$) having $`M_F=m_{F,1}+m_{F,2}=4`$13. Because of their different spin configurations, the atoms and molecules generally have different magnetic moments and the difference depends on magnetic field. Thus $`ϵ`$ depends on magnetic field and the degree of atom–molecule coupling is magnetically tunable. The energy difference between the free atoms and the bound molecules is plotted in Fig. 1A. This behavior of the bound-state energy also causes a resonance in the scattering length, $`a`$, which is shown in Fig. 1B. The scattering length characterizes the mean-field interaction energy of a BEC.

When the magnetic field is tuned to values near the Feshbach resonance, theory predicts coherent oscillations between the atomic and molecular states, but there is significant disagreement on the conversion fraction and the coherence properties16-21.

In experiments with a sodium BEC, Stenger et al.22 observed that inelastic losses were dramatically enhanced when they ramped the magnetic field across the Feshbach resonance. We observed similar results for 85Rb, but with lower rates23,24. It is likely that the formation of molecules played a role in the loss, but there was no experimental evidence for the presence of molecules and the results followed a loss-rate dependence on time. More recently, we measured the time dependence of the loss in an 85Rb BEC by applying controlled magnetic-field pulses toward but not across the Feshbach resonance25. We observed the surprising result that under some conditions, shorter, more rapid pulses actually led to more loss than longer, slower pulses that spent more time near the resonance. The time dependence of the loss was suggestive of a nonadiabatic mixing of states, with the only states within a reasonable energy range being the normal atomic BEC state and the nearby bound molecular state.

In this work we show that much of the loss is likely due to the coherent mixing of atomic and molecular states. To create a superposition and probe its coherence, we applied two short magnetic-field pulses toward the Feshbach resonance, separated by a “free evolution” time during which the magnetic field was held at a constant value some distance from the resonance. We measured the number of atoms in the condensate as a function of time between the two pulses for various values of the steady-state magnetic field between the pulses. We observed dramatic oscillations in the number of atoms remaining in the atomic BEC at frequencies corresponding to the energy splitting between the molecular and the atomic states.

Experimental methods. The apparatus has been described in detail elsewhere23,25. We first created 85Rb condensates23 typically containing 16,500 atoms, with fewer than 1,000 uncondensed thermal atoms. The initial number $`N_{init}`$ fluctuated from shot to shot by $``$500 atoms ($`3`$% number noise). After producing the condensate at a field of $``$162 G, we ramped the magnetic field adiabatically to $``$166 G, corresponding to an initial scattering length $`a_{init}10a_0`$, where $`a_0=0.053`$ nm. The spatial distribution of the atoms was Gaussian with a peak atom density of $`n_0=5.4\times 10^{13}`$ cm-3, and the trap frequencies were (17.4$`\times `$17.4$`\times `$6.8) Hz. After preparing the condensate we applied a selected fast magnetic-field pulse sequence by sending an appropriate time-dependent current through an auxiliary magnetic-field coil25. A typical pulse sequence is shown in Fig. 2. It is composed of two nearly identical short trapezoidal pulses separated by a region of constant (but adjustable) magnetic field. Upon completion of the fast-pulse sequence in Fig. 2, we ramped the magnetic field from $``$166 G to $``$157 G in 5 ms and held at that field for an additional 7 ms to allow the repulsive mean-field energy to expand the condensate. Then we turned off the magnetic trap and used destructive absorption imaging 12.8 ms later to observe the atomic condensate and measure the number of remaining atoms26. This detection scheme was neither sensitive to atoms with kinetic energies larger than $`2\mu `$K nor to atoms in off-resonant molecular states. We determined the value of the magnetic field between the pulses, $`B_{evolve}`$, by measuring the resonance frequency for transitions from the $`F=2`$, $`m_F=2`$ to the $`F=2`$, $`m_F=1`$ spin state by applying a 10 $`\mu `$s RF pulse to a trapped cloud of atoms25.

Burst and remnant oscillations. As we observed for single pulses toward the Feshbach resonance25, there were two distinct components of atoms observed in the absorption images and a third “missing” component that we could not detect. One of the observed components was a cold remnant BEC which was not noticeably heated or excited by the fast-pulse sequence, while the other component was a relatively hot ($``$150 nK) “burst” of atoms that remained magnetically trapped during the BEC expansion time. Using a variational approach27 to model the mean-field expansion that we applied to the BEC remnant to measure its number, $`N_{remnant}`$, we found that we should impart $`3`$ nK worth of energy to the remnant before imaging. This estimate agrees well with the expansion velocity that we observed after the trap turn-off. Thus the remnant BEC was nearly $`50\times `$ colder than the burst.

The missing component contained atoms that were in the initial sample but were not detected after the trap turn-off. To find the number of atoms in the remnant BEC and the number of burst atoms, we allowed the magnetic trap to “focus” the burst cloud before imaging28. A typical image is shown in Fig. 3. We fit the focussed burst (which had a much larger spatial extent than the remnant) with a two-dimensional Gaussian surface, excluding the central region of the image that contained the remnant. This fit yielded the number of burst atoms, $`N_{burst}`$. Subtracting this fit from the image and performing a pixel-by-pixel sum of the central region of the image gave $`N_{remnant}`$.

$`N_{remnant}`$ versus $`t_{evolve}`$ is plotted in Fig. 4 for two different values of $`B_{evolve}`$. The number clearly oscillates. Changing the value of $`B_{evolve}`$ affected the oscillation frequency dramatically (note the change in scale from Fig. 4A to Fig. 4B). After only pulse #1 and the subsequent constant field but with no pulse #2, $`N_{remnant}`$ showed no variation except for a slow decay consistent with the loss rate expected for a single pulse to that field25.

We have taken data similar to those in Fig. 4 for a variety of different $`B_{evolve}`$ values. As in Fig. 4, we fit each curve to the function $`y=y_0+A\mathrm{exp}(t/\tau )\mathrm{sin}(2\pi \nu t+\varphi )`$ to find the oscillation frequency $`\nu `$ and decay time constant $`\tau `$. The measured frequencies are plotted versus $`B_{evolve}`$ in Fig. 5 along with theoretical predictions for the bound-state energy relative to the atomic state.

In the regime where the scattering length is much larger than the radius of the interatomic potential well, the bound state energy for an arbitrary attractive potential can be approximated by $`ϵ=\mathrm{}^2/ma^2`$ 29. $`\mathrm{}`$ is Plank’s constant divided by $`2\pi `$, $`m`$ is the atomic mass, and $`a`$ is the scattering length. The same equation relates the bound state energy to the effective scattering length, which is calculated from the Feshbach resonance parameters through the relation $`a=a_{bg}\times (1\frac{\mathrm{\Delta }}{BB_0})`$, where $`a_{bg}`$ is the background scattering length, $`\mathrm{\Delta }`$ is the width of the Feshbach resonance, and $`B_0`$ is the resonant magnetic field30.

The quantity $`|ϵ|/h`$ is plotted with no adjustable parameters in Fig. 5. The measured oscillation frequencies are in excellent agreement with this simple model over the range of magnetic fields where the model is expected to be valid. The theoretical results found with a much more sophisticated coupled–channels scattering calculation14 in Fig. 5 are in excellent agreement with the data over the entire range.

The fact that the oscillations occurred at exactly the frequency corresponding to the bound-state energy clearly indicates that we are creating a coherent superposition of atoms and molecules with the sudden magnetic-field pulses11,12. Although we do not have a detailed understanding of how the field pulses couple atoms and molecules, by choosing the shapes of the perturbing pulses such that a single pulse results in roughly 50% loss, we observe high-contrast oscillations in the number of atoms in the atomic BEC.

From the amplitude of the oscillations, one can put a lower bound on the number of molecules being created. Take, for example, the data in Fig. 4A. The amplitude of the atom oscillations was 1800(300) atoms. Assuming the fringes are coming from interference with molecules, there must be at least 1800/2 = 900(200) molecules on average. Assuming that the missing atoms are molecules that we fail to convert back into atoms gives an upper bound of 3200(100) molecules for the conditions of Fig. 4A.

The damping time for the oscillations, $`\tau _{decay}`$, was more difficult to measure with high precision than the oscillation frequency. To within our measurement precision, $`\tau _{decay}`$ did not depend on $`B_{evolve}`$, but our uncertainties in $`\tau _{decay}`$ were as large as 100% for some fields. We had the highest precision measurements for frequencies around 200 kHz where the oscillation period was long compared to our experimental timing jitter but short compared to $`\tau _{decay}`$. At frequencies near 200 kHz, we measured $`\tau _{decay}=38(8)\mu `$s for $`n_0=1.3\times 10^{14}`$ cm-3 and $`\tau _{decay}=91(33)\mu `$s when we decreased $`n_0`$ to $`1.1\times 10^{13}`$ cm-3.

$`N_{burst}`$ also had interesting dependencies. For the conditions under which most of the data were collected, the burst contained $``$5000 atoms on average, which is $``$30% of $`N_{init}`$. $`N_{burst}`$ depended on density and varied from one-half of the atoms lost from the condensate for our typical peak density of $`n_0=5.4\times 10^{13}`$ cm-3 to nearly all of the atoms lost from the condensate for $`n_0=1.1\times 10^{13}`$ cm-3. $`N_{burst}`$, $`N_{remnant}`$, and total number of atoms detected are plotted in Fig. 6 for $`B_{evolve}`$ = 159.84(2) G and $`n_0=1.1\times 10^{13}`$ cm-3 ($`5\times `$ lower density than was used for the data shown in Fig. 4). All three components oscillated at the same frequency. The burst oscillation lagged behind the remnant oscillation by 155(4). Since the relative phase shift is nearly 180, the oscillation amplitude for the total number was smaller than either the burst or the remnant oscillation amplitudes. The relative phase depended sensitively on the fall time of pulse #2. For example, when we increased the fall time from 11 $`\mu `$s to 159 $`\mu `$s, the burst oscillation then lagged behind the remnant oscillation by 68(7) and the peak-to-peak amplitude of the total number oscillation was 5600(400).

For the conditions of Fig. 6, $`N_{init}`$ = 17,100 exceeded the time-averaged total number of atoms counted after the pulse sequence by 8(3)% on average. For the higher-density measurements in Fig. 4, 39(4)% of the atoms were missing. Experiments with longer pulses #1 and #2 also had a higher fraction of missing atoms. For example, when we used 50 $`\mu `$s pulses with $`n_0=5.4\times 10^{13}`$ cm-3, 56(3)% were missing.

We have carried out double-pulse measurements with a variety of widths and amplitudes for pulses #1 and #2 and a variety of different densities and initial magnetic fields. Although the oscillation frequency was unchanged, the phase, contrast, and damping of the oscillations did vary. The contrast was very sensitive to the pulse length, and was lower for longer pulses that created more missing atoms. Defining the contrast as the oscillation amplitude divided by the time averaged number of remnant atoms detected, we observed an optimum contrast of 0.42(7) for 15 $`\mu `$s pulses to 156.6(1) G. A single such pulse removed about half of the atoms from the BEC. When the pulse length was comparable to $`\tau _{decay}`$, the contrast was reduced by about a factor of two. Under those conditions, $``$3/4 of the atoms were lost after pulse #1. The phase was shifted, but we did not observe a change in the contrast when we varied the amplitudes of pulses #1 and #2 from $`B`$ = 156.6(1) G (2,400 $`a_0`$) to $`B`$ = 155.1(1) G (24,000 $`a_0`$). The contrast did depend on the intermediate level, however, and was reduced for $`B_{evolve}`$ values closest to the resonance, for which the magnetic-field jumps between $`B_{evolve}`$ and pulses #1 and #2 were shortest.

We also looked for a temperature dependence of both the damping and the frequency at $``$200 KHz and did not see any. The high-temperature data was much noisier than the data for pure condensates, due to unexplained enhanced noise in the number of thermal atoms after the magnetic-field pulse, but when the initial thermal fraction was increased from $`<`$5% to 30%, the data still fit to oscillations with frequency, amplitude, and damping consistent with what was observed with low temperature data.

Conclusions and Outlook. Our interpretation of our observations is that the first magnetic-field pulse provides a sufficiently rapid perturbation to result in nonadiabatic mixing between atomic and molecular states. The superposition then evolves according to the energy difference between the states, which is determined by the magnetic field during the free evolution stage, $`B_{evolve}`$. The second pulse mixes atom and molecule states again, such that the final state of the system depends on the relative phase of atomic and molecular fields at the time of the second pulse. This is somewhat analogous to Ramsey’s method of separated oscillating fields33. Under very limited conditions ($`a`$ near 1700 $`a_0`$), we could also observe Rabi-like oscillations with a single pulse towards the Feshbach resonance. This narrow window results from the conflicting needs for both strong coupling and condensate loss time25 long compared to a Rabi oscillation period.

After pulse #2, a fraction of the coherent molecular component is converted into the energetic but still spin-polarized burst atoms through a yet to be determined process. Another mystery is the missing atoms. Are they molecules that are not converted back into atoms and are not detected in the burst or the remnant signals? If so, why do we not see them as atoms after the field is turned off and the corresponding molecular state is no longer bound? Why are there fewer missing atoms for lower-density condensates and quicker pulses towards the Feshbach resonance? What is the actual conversion efficiency from atoms to molecules and how could we maximize it? Very near the Feshbach resonance, the molecular state has a magnetic moment nearly the same as that of the free atoms, and hence will remain magnetically trapped. A major remaining question concerns the nature of the molecules. Could they be considered a molecular BEC? Clearly there is much to be learned about this curious system.

* Wynar, R. H., Freeland, R. S., Han, D. J., Ryu, C. & Heinzen, D. J. Molecules in a Bose-Einstein condensate. Science 287, 1016-1019 (2000).
* Heinzen, D. J., Wynar, R., Drummond, P. D. & Kheruntsyan, K. V. Superchemistry: dynamics of coupled atomic and molecular Bose-Einstein condensates. Phys. Rev. Lett. 84, 5029-5033 (2000).
* Anglin, J. R. & Vardi, A. Dynamics of a two-mode Bose-Einstein condensate beyond mean-field theory. Phys. Rev. A 64, 013605/1-9 (2001).
* Cusack, B. J., Alexander, T. J., Ostrovskaya, E. A. & Kivshar, Y. S. Existence and stability of coupled atomic-molecular Bose-Einstein condensates. Phys. Rev. A 65, 013609/1-4 (2001).
* Calsamiglia, J., Mackie, M. & Suominen, K. Superposition of macroscopic numbers of atoms and molecules. Phys. Rev. Lett. 87, 160403-1 (2001).
* Drummond, P. D., Kheruntsyan, K. V., Heinzen, D. J. & Wynar, R. H. Stimulated Raman adiabatic passage from an atomic to a molecular Bose-Einstein condensate. (available at http://lanl.arxiv.org/abs/cond-mat/0110578) 1-16 (2002).
* McKenzie, C. et al. Photoassociation of Sodium in a Bose-Einstein Condensate. Phys. Rev. Lett. 88, 120403/1-4 (2001).
* Tiesinga, E., Moerdijk, A., Verhaar, B. J. & Stoof, H. T. C. Conditions for Bose-Einstein condensation in magnetically trapped atomic cesium. Phys. Rev. A 46, R1167-R1170 (1992).
* Tiesinga, E., Verhaar, B. J. & Stoof, H. T. C. Threshold and resonance phenomena in ultracold ground-state collisions. Phys. Rev. A 47, 4114-4122 (1993).
* Moerdijk, A. J., Verhaar, B. J. & Axelson, A. Resonances in ultracold collisions of 6Li, 7Li, and 23Na. Phys. Rev. A 51, 4852-4861 (1995).
* van Abeelen, F. A. & Verhaar, B. J. Time-dependent Feshbach resonance scattering and anomalous decay of a Na Bose-Einstein condensate. Phys. Rev. Lett. 83, 1550-1553 (1999).
* Mies, F. H., Tiesinga, E. & Julienne, P. S. Manipulation of Feshbach resonances in ultracold atomic collisions using time-dependent magnetic fields. Phys. Rev. A 61, 022721/1-17 (2000).
* van Abeelen, F. A., Heinzen, D. J. & Verhaar, B. J. Photoassociation as a probe of Feshbach resonances in cold-atom scattering. Phys. Rev. A 57, R4102-R4105 (1998).
* The coupled–channels calculations were kindly provided by Servaas Kokkelmans and Chris Greene. The calculations used the best estimates of the Feshbach resonance parameters found by combining the results of three high-precision experiments. The determination of the parameters and the construction of the potentials is described in detail by van Kempen et al.15
* van Kempen, E. G. M., Kokkelmans, S. J. J. M. F., Heinzen, D. J. & Verhaar, B. J. Interisotope determination of ultracold rubidium interactions from three high-precision experiments. Phys. Rev. Lett. 88, 093201/1-4 (2002).
* Timmermans, E., Tommasini, P., Hussein, M. & Kerman, A. Feshbach resonances in atomic Bose-Einstein condensates. Phys. Rep. 315, 199-230 (1999).
* Timmermans, E., Tommasini, P., Côté, R., Hussein, M. & Kerman, A. Rarified liquid properties of hybrid atomic-molecular Bose-Einstein condensates. Phys. Rev. Lett. 83, 2691-2691 (1999).
* Drummond, P. D., Kheruntsyan, K. V. & He, H. Coherent molecular solitons in Bose-Einstein condensates. Phys. Rev. Lett. 81, 3055-3058 (1998).
* Holland, M., Park, J. & Walser, R. Formation of pairing fields in resonantly coupled atomic and molecular Bose-Einstein condensates. Phys. Rev. Lett. 86, 1915-1918 (2001).
* Góral, K., Gajda, M. & Rza̧żewski, K. Multimode dynamics of a coupled ultracold atomic-molecular system. Phys. Rev. Lett. 86, 1397-1401 (2001).
* Vardi, A., Yurovsky, V. A. & Anglin, J. R. Quantum effects on the dynamics of a two-mode atom-molecule Bose-Einstein condensate. Phys. Rev. A 64, 063611/1-5 (2001).
* Stenger, J. et al. Strongly enhanced inelastic collisions in a Bose-Einstein condensate near Feshbach resonances. Phys. Rev. Lett. 82, 2422-2425 (1999).
* Cornish, S. L., Claussen, N. R., Roberts, J. L., Cornell, E. A. & Wieman, C. E. Stable 85Rb Bose-Einstein condensates with widely tunable interactions. Phys. Rev. Lett. 85, 1795-1798 (2000).
* Claussen, N. R., Cornish, S. L., Roberts, J. L., Cornell, E. A. & Wieman, C. E. in Atomic Physics 17, (ed. Arimondo, E., DeNatale, P., & Inguscio, M.) 325-336 (American Institute of Physics, New York, 2001).
* Claussen, N. R., Donley, E. A., Thompson, S. T. & Wieman, C. E. Microscopic Dynamics in a Strongly Interacting Bose-Einstein Condensate. in press (available at http://lanl.arxiv.org/abs/cond-mat/0201400) 1-4 (2002).
* Roberts, J. L. et al. Controlled collapse of a Bose-Einstein condensate. Phys. Rev. Lett. 86, 4211-4214 (2001).
* Pérez-García, V. M., Michinel, H., Cirac, J. I., Lewenstein, M. & Zoller, P. Dynamics of Bose-Einstein condensates: variational solutions of the Gross-Pitaevskii equations. Phys. Rev. A 56, 1424-1432 (1997).
* Donley, E. A. et al. Dynamics of collapsing and exploding Bose-Einstein condensates. Nature 412, 295-299 (2001).
* Sakurai, J. J. Modern Quantum Mechanics (Addison-Wesley, Reading, Massachusetts, 1994).
* Rethermalization measurements in 85Rb thermal clouds yield values of $`B_0`$ = 154.9(4) G and $`\mathrm{\Delta }`$ = 11.0(4) G (31). The best estimate of $`a_{bg}`$ is an estimate found by mass scaling spectroscopic measurements of the four highest-lying bound states of 87Rb15, which gives $`a_{bg}=450(3)a_0`$32.
* Roberts, J. L. et al. Improved characterization of elastic scattering near a Feshbach resonance in 85Rb. Phys. Rev. A 64, 024702/1-3 (2001).
* Kokkelmans, S. J. J. M. F. Private Communication.
* Ramsey, N. F. A molecular beam resonance method with separated oscillating fields. Phys. Rev. 78, 695-699 (1950).

Acknowledgements

We would like to acknowledge contributions from E. A. Cornell and the JILA quantum gas collaboration. We are particularly grateful to C. H. Greene and S. J. J. M. F. Kokkelmans for providing us with the coupled-channel scattering calculations presented in Fig. 4 and to L. Pitaevskii for numerous fruitful discussions. S. T. T. acknowledges the support of an ARO-MURI Fellowship. This work was also supported by ONR and NSF.

Correspondence and requests for materials should be addressed to N.R.C.

(e-mail:nclausse@jilau1.colorado.edu.)


text:
# Epitaxial Growth of La1/3Sr2/3FeO3 thin films by laser ablation

## Abstract

We report on the synthesis of high quality La1/3Sr2/3FeO3 (LSFO) thin films using the pulsed laser deposition technique on both SrTiO3 (STO) and LaAlO3 (LAO) substrates (100)-oriented. From X-Ray diffraction (XRD) studies, we find that the films have an out-of-plane lattice parameter around 0.3865nm, almost independent of the substrate (i.e. the nature of the strains). The transport properties reveal that, while LSFO films deposited on STO exhibit an anomaly in the resistivity vs temperature at $`180K`$ (corresponding to the charge-ordered transition and associated with a transition from a paramagnetic to an antiferromagnetic state), the films grown on LAO display a very small magnetoresistance behavior and present an hysteresis around $`270K`$ under the application of a $`4T`$ magnetic field. The changes in transport properties between both substrates are discussed and compared with the corresponding single crystals.

The transition metal oxides (TMO) have been extensively studied in the past years, in particular due to the metal-insulator (MI) transition . Among the different compounds, the manganites RE1-xAxMnO3 (RE=rare earths, A=alkaline earths) have received a great interest these last years due to their colossal magnetoresistance (MR) properties . An interesting feature of these materials is the charge-ordering phenomena  i.e. the ordering of cations of different charges (Mn3+/Mn4+ for example) on specific lattice sites. This characteristic can be seen in others compounds such as nickelates  or layered manganites . In fact, it has also been recently observed in others perovskites such as La1/3Sr2/3MnO3 (LSFO) , which has been first studied by Waugh et al. . Even if more studies appear very recently on such materials , there are vey few reports on thin films growth and to our knowledge, only for the particular composition SrFeO3-δ . Interestingly, theses materials show properties close to the manganites and charge disproportionation has also been investigated in the past . Moreover, they are suitable candidates for MR properties . The synthesis of charge-ordered (CO) manganite thin films is difficult owing to the distortion of the lattice induce by the substrate  and usually, the properties of the CO thin films are different from the bulk. For example, in the case of Pr0.5Ca0.5MnO3, the CO state can not been completely established. This means that the CO state is weaken due to the substrate-induce strains because it is impossible to accomodate the quite large changes of structural parameters when occuring below the CO transition ($`T_{CO}`$) .

In an effort of understanding the particular substrate-effects of the CO oxide compounds, it is interesting to look at others compositions. La1/3Sr2/3FeO3 is such an example. For these reasons, we have synthesized La1/3Sr2/3FeO3 thin films on two types of substrates that induce compression or tensile strains. We measured the structural lattice parameters and transport properties of the films. Our results are report in this communication.

Thin films of LSFO were grown using the pulsed laser deposition technique. The target used has a nominal composition of La1/3Sr2/3FeO3. In the bulk form, LSFO has a rhombohedral structure ($`R\overline{3}c`$) with a lattice constant $`a_R=0.547nm`$ and an edge angle $`a_R=60.1^{}`$ at room temperature . In fact, the average structure can be considered as a cubic perovskite slightly distorted with a parameter close to $`a2a_P2\times 0.387nm=0.774nm`$ (where $`a_P`$ being the ideal parameter of the cubic perovskite). Thus, we expect to be able to grow a thin film of this composition directly on a perovskite substrate. For facilitating the reading of this paper, we keep the simple cubic cell notation (index with C). Substrates used were (100)-SrTiO3 (STO, cubic with $`a=0.3905nm`$). Also, (100)-LaAlO3 substrates (LAO, pseudocubic with $`a=0.3788nm`$) were utilized in order to compare the effect of tensile or compressive stress. The substrates were kept at a constant temperature around $`710{}_{}{}^{}C`$ during the deposition which was carried out at a pressure of $`200mTorr`$ of flowing oxygen. After deposition, the samples were slowly cooled to room temperature at a pressure of $`500Torr`$ of oxygen. The deposition rate is $`4Hz`$ and the energy density is close to $`2J/cm^2`$. Further details of the targets preparation and the deposition procedure are given elsewhere . The structural study was done by X-Ray diffraction (XRD) using a Seifert XRD 3000P for the $`\mathrm{\Theta }2\mathrm{\Theta }`$ scans and a X’Pert Phillips for the in-plane measurements (Cu, K$`\alpha `$ , $`\lambda `$=0.15406nm). The composition of the film was checked and corresponds to the nominal one in the limit of the accuracy. Direct current resistivity was measured by a four-probe method under magnetic fields up to 7T using a Quantum Design PPMS and magnetization was recorded using a Quantum Design MPMS SQUID magnetometer as a function of the temperature in the range 4-300K.

Fig.1a shows the $`\mathrm{\Theta }2\mathrm{\Theta }`$ scan for an as-grown film deposited on STO substrate. Two diffraction peaks can be seen corresponding to an out-of-plane parameter of 0.3865nm. These two peaks can be indexed, based on the cubic perovskite, as the (100)C and the (200)C reflections. Note that the high intensity and the sharpness of the peaks, reveal a high quality of the growth which is confirmed by the low value ($`0.3^{}`$) of the full-width-at-half-maximum (FWHM) of the $`\omega `$-scan recorded around the (002)C (see inset of Fig.1a). Data used to determine the in-plane lattice parameters of LSFO thin film were obtained from asymmetric XRD scans of the STO (202) and (220) reflections. These scans detected the (202)C and (220)C of LSFO. The unit cell is found to be tetragonal with $`a=0.3882nm`$ and $`c=0.3865nm`$. These values are close to the bulk value, referring to the cubic unit cell ($`a0.387nm`$) . In fact, the out–of-plane lattice parameter decreases slightly and the in-plane lattice parameter increases lightly confirming that the LSFO film is under tensile stress on STO substrate, keeping the volume of the cell almost constant ($`58.24\AA ^3`$ in the film to be compared with $`57.96\AA ^3`$ in the corresponding bulk material). Fig.1b shows a $`\mathrm{\Phi }`$-scan obtained around the (202)C reflection. Four peaks separated from $`90^{}`$, are visible and correspond to a four-fold symmetry. The sharp peaks at $`90^{}`$ intervals indicate a perfect in-plane alignment with the substrate. Details investigations of the structure and the microstructure are in progress using high resolution electron microscopy and will be published elsewhere. The $`\mathrm{\Theta }2\mathrm{\Theta }`$ scan for an as-grown film deposited on LAO substrates is presented in Fig.2. As in the case of STO, two diffraction peaks are clearly visible close to those of the substrate, corresponding to an out-of-plane parameter of $`0.386nm`$. Surprisingly, this value is very close to the one obtained on STO indicating that the strains seem not to play an important role on the structure (or at least on the lattice parameters) of these compounds. Even if lattice parameters are similar for both substrates, the quality of the film are different. This is evidenced by the full width at half-maximum (FWHM) of the $`\omega `$-scan recorded around the (002)C (see inset of Fig.2). On LAO, the value is higher ($`0.4^{}`$) on this substrate. The rocking curves measurements characterize informations on the angular distribution of the LSFO crystallites in the film. Thus, the narrow value on STO as compared to LAO, shows that the $`c`$-axis parameter of LSFO films on LAO has a spread mosaicity, i.e. there are not perfectly parallel to each others.

We present in Fig.3 the temperature dependencies of the resistivity as well as the magnetization for a LFSO film grown on STO. The resistivity at room temperature is low ($`10^4\mathrm{\Omega }.cm`$) but it is still about one order of magnitude higher than in the bulk . When the temperature is decreasing, the resistivity is increasing almost gradually with a semiconductorlike behavior. An anomaly around $`180K`$ is observed and corresponds to the charge-ordered transition $`T_{CO}`$. At this temperature, the magnetization shows a cusp which is characteristic of an antiferromagnetic transition. However, at low temperature the magnetization value is still very high but most probably a result of the substrate component. Different features can be seen from these datas as compared with the single crystals . First, the cusp in magnetization is weaker in the film and second, there is no an abrupt change in resistivity as seen in LSFO single crystals. This is not surprising because the charge-ordering transition is never as pronounced in thin films , even if the structure shows an ordering at low temperature  and this is due to the presence of substrate which does not allowed the change of lattice parameters below the CO transition observed in the bulk. One can also notice that $`T_{CO}`$ is $`20K`$ lower than the in the bulk ($`200K`$) . The first explanation of the decreasing of the $`T_{CO}`$ is that, it is often difficult to reach the optimized properties in thin films without a complex growth process , mostly due to the substrate-induced strain which bends the structure of the film. Another possibility might comes for the oxygen stoechiometry since the volume of the cell of the film is increasing of $`0.3\AA ^3`$. According to Dann et al., it would correspond to an increase of the Sr content. Equivalently, this would correspond to an increasing of the oxygen . In our LSFO film, the changes of lattice parameter confirm the tensile stress of the film and an increase of the oxygen content led to a decrease of both lattice parameters (in-plane and out-of-plane) . Since in our case, only one parameter is increasing, we think that the oxygen content is not the reason of such a behavior.

Fig.4 shows the $`\rho (T)`$ without field and with a $`7T`$ magnetic field for the LSFO film deposited on LAO. We can see that the film displays an insulating behavior characteristic of the CO compound. However, in this LSFO on LAO, we can notice that the film exhibit a small magnetoresistance effect under a $`7T`$ magnetic field. Moreover, an anomaly around $`260K`$ with a thermal hysteresis of few kelvins, are observed. These two features correspond to a transition which is, in fact, close and higher to the $`T_{CO}`$ transition.

These results indicate that the CO state is weaker when deposited on STO rather than on LAO since the $`T_{CO}`$ is shifted to higher temperature in the LAO case. In others words, the charge-ordered state becomes more stable under compressive strain. Such feature as been observed recently in the case of manganite thin films where the ferromagnetism is enhanced under tensile stress  (or equivalently the CO state becomes less stable under tensile stress). The charge-ordered state seems to behave in the same way with Mn and Fe based compounds. Further compositions need to be investigated in the future to confirm theses results.

In conclusion, we have investigated the structural, transport and magnetic properties of La1/3Sr2/3FeO3 thin films grown by the pulsed laser deposition technique. Highly crystallized films were obtained on both substrates SrTiO3 and LaAlO3. We have seen that the out-of-plane lattice parameter is almost independent of the type of strains. The films show an anomaly in $`\rho (T)`$ and $`M(T)`$ close to the corresponding charge-ordered transition observed in the bulk material. However, this transition is lower in the case of STO and higher in the case of LAO, as compared to the bulk compound confirming that a charge-oreder state in less stable under tensile stress. We explained these differences by the stress induced by the substrate upon the film.

Figures Captions:

Figure 1a: Room temperature $`\mathrm{\Theta }2\mathrm{\Theta }`$ XRD pattern of LSFO on STO. Peaks labeled with the index S refer to the substrate peaks. The inset is the rocking curve ($`\omega `$-scan) of (002)C reflection of the LSFO film.

Figure 1b: $`\mathrm{\Phi }`$-scan of the (202)C family peaks in LSFO film showing a very good in-plane orientation of the film.

Figure 2: Room temperature $`\mathrm{\Theta }2\mathrm{\Theta }`$ XRD pattern of LSFO on LAO. Peaks labeled with the index S refer to the substrate peaks. The inset is the rocking curve ($`\omega `$-scan) of (002)C reflection of the LSFO film.

Figure 3: Temperature dependence of the resistivity $`(\rho )`$ and magnetization $`(M)`$ at $`\mu _0H=0.5T`$ for a LSFO film on STO. No MR was observed with a 7T magnetic field. The magnetic field is applied perpendicular to the surface of the substrate.

Figure 4: Temperature dependence of the resistivity $`(\rho )`$ for a LSFO on LAO under different applied magnetic field. Note the small magnetoresistance effect. The inset is magnified resistivity curve in the range $`240300K`$ showing a clear phase transition around $`270K`$. The magnetic field is applied perpendicular to the surface of the substrate.