Structural insights into fibronectin type III domain-mediated signaling

The alternatively spliced type III extradomain B (EIIIB) of fibronectin (FN) is expressed only during embryogenesis, wound healing and tumorigenesis. The biological function of this domain is unclear. We describe here the first crystal structure of the interface between alternatively spliced EIIIB and its adjacent FN type III domain 8 (FN B-8). The opened CC' loop of EIIIB, and the rotation and tilt of EIIIB allow good access to the FG loop of FN-8, which is normally hindered by the CC' loop of FN-7. In addition, the AGEGIP sequence of the CC' loop of EIIIB replaces the NGQQGN sequence of the CC' loop of FN-7. Finally, the CC' loop of EIIIB forms an acidic groove with FN-8. These structural findings warrant future studies directed at identifying potential binding partners for FN B-8 interface, linking EIIIB to skeletal and cartilaginous development, wound healing, and tumorigenesis, respectively.     

Predicting antibody hypervariable loop conformations. II: Minimization and molecular dynamics studies of MCPC603 from many randomly generated loop conformations

We describe a method for predicting the conformations of loops in proteins and its application to four of the complementarity determining regions [CDRs] in the crystallographically determined structure of MCPC603. The method is based on the generation of a large number of randomly generated conformations for the backbone of the loop being studied, followed by either minimization or molecular dynamics followed by minimization starting from these random structures. The details of the algorithm for the generation of the loops are presented in the first paper in this series (Shenkin et al. [submitted]). The results of minimization and molecular dynamics applied to these loops is presented here. For the two shortest CDRs studied (H1 and L2, which are five and seven amino acids long), minimizations and dynamics simulations which ignore interactions of the loop amino acids beyond the carbon beta replicate the conformation of the crystal structure closely. This suggests that these loops fold independently of sequence variation. For the third CDR (L3, which is nine amino acids), those portions of the CDR near its base which are hydrogen bonded to framework are well replicated by our procedures, but the top of the loop shows significant conformational variability. This variability persists when side chain interactions for the MCPC603 sequence are included. For a fourth CDR (H3, which is 11 amino acids long), new low-energy backbone conformations are found; however, only those which are close to the crystal are compatible with the sequence when side chain interactions are taken into account. Results from minimization and dynamics on single CDRs with all other CDRs removed are presented. These allow us to explore the extent to which individual CDR conformations are determined by interactions with framework only.     

Mechanism of Tet repressor induction by tetracyclines: length compensates for sequence in the alpha8-alpha9 loop

Natural Tet repressor (TetR) variants are alpha-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the alpha8-alpha9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 10(5) M(-1) for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 x 10(9) M(-1) and 8.0 x 10(9) M(-1), about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length-to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction.     

Triggering loops and enzyme function: identification of loops that trigger and modulate movements

Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. beta1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.     

Modeling protein loops using a phi i + 1, psi i dimer database.

We present an automated method for modeling backbones of protein loops. The method samples a database of phi i + 1 and psi i angles constructed from a nonredundant version of the Protein Data Bank (PDB). The dihedral angles phi i + 1 and psi i completely define the backbone conformation of a dimer when standard bond lengths, bond angles, and a trans planar peptide configuration are used. For the 400 possible dimers resulting from 20 natural amino acids, a list of allowed phi i + 1, psi i pairs for each dimer is created by pooling all such pairs from the loop segments of each protein in the nonredundant version of the PDB. Starting from the N-terminus of the loop sequence, conformations are generated by assigning randomly selected pairs of phi i + 1, psi i for each dimer from the respective pool using standard bond lengths, bond angles, and a trans peptide configuration. We use this database to simulate protein loops of lengths varying from 5 to 11 amino acids in five proteins of known three-dimensional structures. Typically, 10,000-50,000 models are simulated for each protein loop and are evaluated for stereochemical consistency. Depending on the length and sequence of a given loop, 50-80% of the models generated have no stereochemical strain in the backbone atoms. We demonstrate that, when simulated loops are extended to include flanking residues from homologous segments, only very few loops from an ensemble of sterically allowed conformations orient the flanking segments consistent with the protein topology. The presence of near-native backbone conformations for loops from five different proteins suggests the completeness of the dimeric database for use in modeling loops of homologous proteins. Here, we take advantage of this observation to design a method that filters near-native loop conformations from an ensemble of sterically allowed conformations. We demonstrate that our method eliminates the need for a loop-closure algorithm and hence allows for the use of topological constraints of the homologous proteins or disulfide constraints to filter near-native loop conformations.     

Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain

The tenth fibronectin type III domain of human fibronectin (FNfn10) is a small, monomeric beta-sandwich protein, similar to the immunoglobulins. We have developed small antibody mimics, 'monobodies', using FNfn10 as a scaffold. We initially altered two loops of FNfn10 that are structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. In order to assess the possibility of utilizing other loops in FNfn10 for target binding, we determined the effects of the elongation of each loop on the conformational stability of FNfn10. We found that all six loops of FNfn10 allowed the introduction of four glycine residues while retaining the global fold. Insertions in the AB and FG loops exhibited very small degrees of destabilization, comparable to or less than predicted entropic penalties due to the elongation, suggesting the absence of stabilizing interactions in these loops in wild-type FNfn10. Insertions in the BC, CD and DE loops, respectively, resulted in modest destabilization. In contrast, the EF loop elongation was highly destabilizing, consistent with previous studies showing the presence of stabilizing interactions in this loop. These results suggest that all loops, except for the EF loop, can be used for engineering a binding site, thus demonstrating excellent properties of the monobody scaffold.     

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.     

A dual function for SecA in the assembly of single spanning membrane proteins in Escherichia coli

The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particle-dependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle- and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecA-independent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.     

The influence of the local sequence environment on RNA loop structures

RNA folding is assumed to be a hierarchical process. The secondary structure of an RNA molecule, signified by base-pairing and stacking interactions between the paired bases, is formed first. Subsequently, the RNA molecule adopts an energetically favorable three-dimensional conformation in the structural space determined mainly by the rotational degrees of freedom associated with the backbone of regions of unpaired nucleotides (loops). To what extent the backbone conformation of RNA loops also results from interactions within the local sequence context or rather follows global optimization constraints alone has not been addressed yet. Because the majority of base stacking interactions are exerted locally, a critical influence of local sequence on local structure appears plausible. Thus, local loop structure ought to be predictable, at least in part, from the local sequence context alone. To test this hypothesis, we used Random Forests on a nonredundant data set of unpaired nucleotides extracted from 97 X-ray structures from the Protein Data Bank (PDB) to predict discrete backbone angle conformations given by the discretized η/θ-pseudo-torsional space. Predictions on balanced sets with four to six conformational classes using local sequence information yielded average accuracies of up to 55%, thus significantly better than expected by chance (17%-25%). Bases close to the central nucleotide appear to be most tightly linked to its conformation. Our results suggest that RNA loop structure does not only depend on long-range base-pairing interactions; instead, it appears that local sequence context exerts a significant influence on the formation of the local loop structure.     

Shortening a loop can increase protein native state entropy

Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all‐atom molecular dynamics simulations to study how gradual shortening a very long or solvent‐exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. Proteins 2015; 83:2137–2146. © 2015 Wiley Periodicals, Inc.     

Achieving Peptide Binding Specificity and Promiscuity by Loops: Case of the Forkhead-Associated Domain

The regulation of a series of cellular events requires specific protein–protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, α helix and β sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.     

Structural and Functional Roles of the Surface-Exposed Loops of the β-Barrel Membrane Protein OmpA from Escherichia coli

The N-terminal domain of the OmpA protein from Escherichia coli, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel beta-barrel whose eight transmembrane beta-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest beta-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.     

Fast de novo discovery of low‐energy protein loop conformations

In the prediction of protein structure from amino acid sequence, loops are challenging regions for computational methods. Since loops are often located on the protein surface, they can have significant roles in determining protein functions and binding properties. Loop prediction without the aid of a structural template requires extensive conformational sampling and energy minimization, which are computationally difficult. In this article we present a new de novo loop sampling method, the Parallely filtered Energy Targeted All‐atom Loop Sampler (PETALS) to rapidly locate low energy conformations. PETALS explores both backbone and side‐chain positions of the loop region simultaneously according to the energy function selected by the user, and constructs a nonredundant ensemble of low energy loop conformations using filtering criteria. The method is illustrated with the DFIRE potential and DiSGro energy function for loops, and shown to be highly effective at discovering conformations with near‐native (or better) energy. Using the same energy function as the DiSGro algorithm, PETALS samples conformations with both lower RMSDs and lower energies. PETALS is also useful for assessing the accuracy of different energy functions. PETALS runs rapidly, requiring an average time cost of 10 minutes for a length 12 loop on a single 3.2 GHz processor core, comparable to the fastest existing de novo methods for generating an ensemble of conformations. Proteins 2017; 85:1402–1412. © 2017 Wiley Periodicals, Inc.     

Identification of neutralizing conformational epitopes on the human papillomavirus type 31 major capsid protein and functional implications

The aim of this study was to characterize the conformational neutralizing epitopes of the major capsid protein of human papillomavirus type 31. Analysis of the epitopes was performed by competitive epitope mapping using 15 anti‐HPV31 and by reactivity analysis using a HPV31 mutant with an insertion of a seven‐amino acid motif within the FG loop of the capsid protein. Fine mapping of neutralizing conformational epitopes on HPV L1 was analyzed by a new approach using a system displaying a combinatorial library of constrained peptides exposed on E. coli flagella. The findings demonstrate that the HPV31 FG loop is dense in neutralizing epitopes and suggest that HPV31 MAbs bind to overlapping but distinct epitopes on the central part of the FG loop, in agreement with the exposure of the FG loop on the surface of HPV VLPs, and thus confirming that neutralizing antibodies are mainly located on the tip of capsomeres. In addition, we identified a crossreacting and partially crossneutralizing conformational epitope on the relatively well conserved N‐terminal part of the FG loop. Moreover, our findings support the hypothesis that there is no correlation between neutralization and the ability of MAbs to inhibit VLP binding to heparan sulfate, and confirm that the blocking of virus attachment to the extracellular matrix is an important mechanism of neutralization.     

Ab initio structure prediction of the antibody hypervariable H3 loop

Antibodies have the capability of binding a wide range of antigens due to the diversity of the six loops constituting the complementarity determining region (CDR). Among the six loops, the H3 loop is the most diverse in structure, length, and sequence identity. Prediction of the three‐dimensional structures of antibodies, especially the CDR loops, is an important step in the computational design and engineering of novel antibodies for improved affinity and specificity. Although it has been demonstrated that the conformation of the five non‐H3 loops can be accurately predicted by comparing their sequences against databases of canonical loop conformations, no such connection has been established for H3 loops. In this work, we present the results for ab initio structure prediction of the H3 loop using conformational sampling and energy calculations with the program Prime on a dataset of 53 loops ranging in length from 4 to 22 residues. When the prediction is performed in the crystal environment and including symmetry mates, the median backbone root mean square deviation (RMSD) is 0.5 Å to the crystal structure, with 91% of cases having an RMSD of less than 2.0 Å. When the prediction is performed in a noncrystallographic environment, where the scaffold is constructed by swapping the H3 loops between homologous antibodies, 70% of cases have an RMSD below 2.0 Å. These results show promise for ab initio loop predictions applied to modeling of antibodies. © 2012 Wiley Periodicals, Inc.     

Sequence codes for extended conformation: a neighbor-dependent sequence analysis of loops in proteins

We performed an extensive sequence analysis on the loops of proteins. By dividing a loop databank derived from the Protein Data Bank into groups, we analyzed the chemical characteristics and the sequence preferences of loops of different lengths and loops connecting different secondary structures in proteins. We found that a large population of loops in our loop databank (94.4%) is either partially or completely surface-exposed. A majority of surface loops in proteins are hydrophilic, whereas the chemical characteristics of interior loops are relatively neutral according to Eisenberg's consensus hydrophobicity scale. As a first step in investigating the intrinsic sequence-structure relationship of loop sequences in proteins, we performed a neighbor-dependent sequence analysis that calculated the effect of the neighboring amino acid type on the loop propensity of residues in loops. This method enhances the statistical significance of residue propensity, thus allowing us to explore the positional preference of amino acids in loops. Our analysis yielded a series of amino acid dyads that showed high preference for loop conformation. The data presented in this study should prove useful for developing potential codes in recognizing loop sequences in proteins.     

Surface loop dynamics in adeno-associated virus capsid assembly

The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.     

New efficient statistical sequence-dependent structure prediction of short to medium-sized protein loops based on an exhaustive loop classification.

A bank of 13,563 loops from three to eight amino acid residues long, representing motifs between two consecutive regular secondary structures, has been derived from protein structures presenting less than 95 % sequence identity. Statistical analyses of occurrences of conformations and residues revealed length-dependent over-representations of particular amino acids (glycine, proline, asparagine, serine, and aspartate) and conformations (alphaL, epsilon, betaPregions of the Ramachandran plot). A position-dependent distribution of these occurrences was observed for N and C-terminal residues, which are correlated to the nature of the flanking regions. Loops of the same length were clustered into statistically meaningful families on the basis of their backbone structures when placed in a common reference frame, independent of the flanks. These clusters present significantly different distributions of sequence, conformations, and endpoint residue Calphadistances. On the basis of the sequence-structure correlation of this clustering, an automatic loop modeling algorithm was developed. Based on the knowledge of its sequence and of its flank backbone structures each query loop is assigned to a family and target loop supports are selected in this family. The support backbones of these target loops are then adjusted on flanking structures by partial exploration of the conformational space. Loop closure is performed by energy minimization for each support and the final model is chosen among connected supports based upon energy criteria. The quality of the prediction is evaluated by the root-mean-square deviation (rmsd) between the final model and the native loops when the whole bank is re-attributed on itself with a Jackknife test. This average rmsd ranges from 1.1 A for three-residue loops to 3.8 A for eight-residue loops. A few poorly predicted loops are inescapable, considering the high level of diversity in loops and the lack of environment data. To overcome such modeling problems, a statistical reliability score was assigned for each prediction. This score is correlated to the quality of the prediction, in terms of rmsd, and thus improves the selection accuracy of the model. The algorithm efficiency was compared to CASP3 target loop predictions. Moreover, when tested on a test loop bank, this algorithm was shown to be robust when the loops are not precisely delimited, therefore proving to be a useful tool in practice for protein modeling.     

Role of the closing base pair for d(GCA) hairpin stability: free energy analysis and folding simulations

Hairpin loops belong to the most important structural motifs in folded nucleic acids. The d(GNA) sequence in DNA can form very stable trinucleotide hairpin loops depending, however, strongly on the closing base pair. Replica-exchange molecular dynamics (REMD) were employed to study hairpin folding of two DNA sequences, d(gcGCAgc) and d(cgGCAcg), with the same central loop motif but different closing base pairs starting from single-stranded structures. In both cases, conformations of the most populated conformational cluster at the lowest temperature showed close agreement with available experimental structures. For the loop sequence with the less stable G:C closing base pair, an alternative loop topology accumulated as second most populated conformational state indicating a possible loop structural heterogeneity. Comparative-free energy simulations on induced loop unfolding indicated higher stability of the loop with a C:G closing base pair by ~3 kcal mol(-1) (compared to a G:C closing base pair) in very good agreement with experiment. The comparative energetic analysis of sampled unfolded, intermediate and folded conformational states identified electrostatic and packing interactions as the main contributions to the closing base pair dependence of the d(GCA) loop stability.