Folding & design最新文献

Background: Small folded polypeptide motifs represent highly simplified systems for theoretical and experimental studies on protein structure and folding. We have recently reported the design and characterization of a metal-ion-independent 23-residue peptide with a ββα structure (BBA1), based on the zinc finger domains. To understand better the determinants of structure for this small peptide, we investigated the conformational role of the synthetic residue 3-(1,10-phenan-throl-2-yl)-L-alanine (Fen) in BBA1.

Results: NMR analysis revealed that replacing the Fen residue of peptide BBA1 by either of the natural amino acids tyrosine (BBA2) or tryptophan (BBA3) resulted in conformational flexibility in the sheet and loop regions of the structure. This conformational ambiguity was eliminated in peptides BBA4 and BBA5 by including charged residues on the exterior of the β hairpin designed to both select against the undesired fold and stabilize the desired structure. The evaluation of two additional peptides (BBA6 and BBA7) provided further insight into the specific involvement of the surface polar residues in the creation of well-defined structure in BBA4 and BBA5. The sequences of BBA5, BBA6 and BBA7 include only one non-standard amino acid (Dproline), which constrains a critical engineered type II′ turn.

Conclusions: Manipulation of residues on the exterior of small ββα motifs has led to the design of 23-residue polypeptides that adopt a defined tertiary structure in the absence of synthetic amino acids, increasing the availability and expanding the potential uses of the BBA motif. The importance of negative design concepts to the creation of structured polypeptides is also highlighted.

Three-stranded coiled coils and three-helix bundles are increasingly being identified in proteins. Design and engineering on the scaffolds of these motifs is a potential route towards combating associated viral infections as well as introducing novel functional sites.

Background: The root mean square deviation (rmsd) between corresponding atoms of two protein chains is a commonly used measure of similarity between two protein structures. The smaller the rmsd is between two structures, the more similar are these two structures. In protein structure prediction, one needs the rmsd between predicted and experimental structures for which a prediction can be considered to be successful. Success is obvious only when the rmsd is as small as that for closely homologous proteins (< 3 å). To estimate the quality of the prediction in the more general case, one has to compare the native structure not only with the predicted one but also with randomly chosen protein-like folds. One can ask: how many such structures must be considered to find a structure with a given rmsd from the native structure?

Results: We calculated the rmsd values between native structures of 142 proteins and all compact structures obtained in the threading of these protein chains over 364 non-homologous structures. The rmsd distributions have a Gaussian form, with the average rmsd approximately proportional to the radius of gyration.

Conclusions: We estimated the number of protein-like structures required to obtain a structure within an rmsd of 6 å to be 10⁴–10⁵ for chains of 60–80 residues and 10¹¹–10¹² structures for chains of 160–200 residues. The probability of obtaining a 6 å rmsd by chance is so remote that when such structures are obtained from a prediction algorithm, it should be considered quite successful.

Background: Antibodies are prototypes of multimeric proteins and consist of structurally similar domains. The two variable domains of an antibody (V_H and V_L) interact through a large hydrophobic interface and can be expressed as covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv fragments after long-term denaturation in guanidinium chloride is known to be slow. In order to delineate the nature of the rate-limiting step, the folding of the scFv fragment of an antibody after short-term denaturation has been investigated.

Results: Secondary structure formation, measured by H/D-exchange protection, of a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium chloride was shown to be multiphasic. NMR analysis shows that an intermediate with significant proton protection is observed within the dead time of the manual mixing experiments. Subsequently, the folding reaction proceeds via a biphasic reaction and mass spectrometry analyses of the exchange experiments confirm the existence of two parallel pathways. In the presence of cyclophilin, however, the faster of the two phases vanishes (when followed by intrinsic tryptophan fluorescence), while the slower phase is not significantly enhanced by equimolar cyclophilin.

Conclusions: The formation of an early intermediate, which shows amide-proton exchange protection, is independent of proline isomerization. Subsequently, a proline cis–trans isomerization reaction in the rapidly formed intermediate, producing ‘non-native’ isomers, competes with the fast formation of native species. Interface formation in a folding intermediate of the scFv fragment is proposed to prevent the back-isomerization of these prolines from being efficiently catalyzed by cyclophilin.

Background: PERB11 is a multicopy polymorphic gene family found in association with HLA Class I genes within the major histocompatibility complex (MHC). Although its function is unknown, PERB11 has sequence similarities to HLA Class I and other related proteins. To explore the possible functional roles for PERB11, homology models have been constructed using both HLA Class I and Class I-like protein structures as templates.

Results: The models show that PERB11.1 appears to have an unusual distribution of charged residues that potentially give the molecule a distinct polarity. Furthermore, a cluster of negatively charged residues in the traditional P2 site may form a novel binding site for a positively charged ligand such as a metal ion or complex. Other charged residues line the floor and walls of the cleft and are able to form salt bridges, reminiscent of the closed cleft of the Class I-like mouse neonatal Fc receptor structure. The closely related PERB11.2 family has a different arrangement of charged residues in the cleft, but these residues are still able to form salt bridges. Unlike HLA Class I, the majority of polymorphic positions in the PERB11 family occur outside the cleft and on the surface of the molecule.

Conclusions: Homology models for PERB11 suggest that the structure is capable of associating with β2 microglobulin or a similar molecule. Furthermore, not all of the potential glycosylation sites suggested by the PERB11 sequences appear viable. Importantly, the models suggest that the molecule has a less accessible cleft than HLA Class I and is not, therefore, able to bind peptides. Other small ligands, including metal ions, might be bound, however.

Metalloprotein properties result from the interplay between coordination requirements of the metal center, protein stability, and modulation of the metal center by the surrounding protein matrix. Simple metal centers, which exercise control over the protein by affecting stability or enzyme activity, have been created by rational design. Complex centers, which require control by the protein matrix, have also been constructed.

Background: It has been observed that single-domain proteins and domains in multidomain proteins favor a chain length in the range 100–50 amino acids. To understand the origin of the favored size, we construct an empirical function for the free energy of unfolding versus the chain length. The parameters in the function are derived by fitting to the energy of hydration, entropy and enthalpy of unfolding of nine proteins. Our energy function cannot be used to calculate the energetics accurately for individual proteins because the energetics also depend on other factors, such as the composition and the conformation of the protein. Nevertheless, the energy function statistically characterizes the general relationship between the free energy of unfolding and the size of the protein.

Results: The predicted optimal number of residues, which corresponds to the maximum free energy of unfolding, is 100. This is in agreement with a statistical analysis of protein domains derived from their experimental structures. When a chain is too short, our energy function indicates that the change in enthalpy of internal interactions is not favorable enough for folding because of the limited number of inter-residue contacts. A long chain is also unfavorable for a single domain because the cost of configurational entropy increases quadratically as a function of the chain length, whereas the favorable change in enthalpy of internal interactions increases linearly.

Conclusions: Our study shows that the energetic balance is the dominant factor governing protein sizes and it forces a large protein to break into several domains during folding.

Background: A common approach to the protein folding problem involves computer simulation of folding using lattice models of amino acid sequences. Key factors for good performance in such models are the correct choice of the temperature and the average interaction energy between residues. In order to push the lattice approach to its limit it is important to have a method to adjust these parameters for optimal folding that is not limited by our ability to successfully simulate folding in a reasonable time.

Results: In this study, we adopt a simple cubic-lattice model and present a method for calculating the free energy of a chain as a function of the number of native contacts. This does not require that we are able to fold the sequence by simulation and it provides a method of estimating the folding transition temperature. For a given set of parameters, the free energy analysis also allows an estimate of foldability. By applying the method to sequences with 27 and 125 residues, we show that optimal folding occurs near the folding transition temperature and at either zero or small negative average interaction energy. We find ourselves able to fold only 125-mers that have significant short-range native contacts.

Conclusions: A free energy analysis during unfolding is a useful tool for the study of foldability and should be applicable to a variety of folding models. In this way we are able to fold some 125-mer designed sequences and our results confirm the finding that short-range contacts contribute to foldability.