Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR.

The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.     

Functionalized oligoanthranilamides: modular and conformationally controlled scaffolds.

This paper describes the use of functionalized oligoanthranilamides as conformationally controlled scaffolds for molecular recognition. Oligomers of anthranilamides are stabilized by the formation of intramolecular six-membered hydrogen bonds in a linear strand conformation. Onto alternate anthranilic acid units, we have attached di- or tripeptide recognition units with the potential to form intramolecular hydrogen bonds to an intercalated peptide strand. Using 1H NMR dilution experiments in CDCl(3), we have observed chemical shift changes that are consistent with the formation of an extended hydrogen bonded sheet dimer. We also demonstrate that the bis-alanine functionalized strands are able to form discrete hydrogen bonded complexes with dipeptide substrates and to bind hexanoyl alanylalanine selectively over its benzyl ester. In the presence of excess hydrogen bond donors and acceptors, the oligoanthranilamide strand retained its linear conformation, pointing to the potential of this modular design as a useful and stable scaffold for molecular recognition studies.     

Beta edge strands in protein structure prediction and aggregation

It is well established that recognition between exposed edges of beta-sheets is an important mode of protein-protein interaction and can have pathological consequences; for instance, it has been linked to the aggregation of proteins into a fibrillar structure, which is associated with a number of predominantly neurodegenerative disorders. A number of protective mechanisms have evolved in the edge strands of beta-sheets, preventing the aggregation and insolubility of most natural beta-sheet proteins. Such mechanisms are unfavorable in the interior of a beta-sheet. The problem of distinguishing edge strands from central strands based on sequence information alone is important in predicting residues and mutations likely to be involved in aggregation, and is also a first step in predicting folding topology. Here we report support vector machine (SVM) and decision tree methods developed to classify edge strands from central strands in a representative set of protein domains. Interestingly, rules generated by the decision tree method are in close agreement with our knowledge of protein structure and are potentially useful in a number of different biological applications. When trained on strands from proteins of known structure, using structure-based (Dictionary of Secondary Structure in Proteins) strand assignments, both methods achieved mean cross-validated, prediction accuracies of approximately 78%. These accuracies were reduced when strand assignments from secondary structure prediction were used. Further investigation of this effect revealed that it could be explained by a significant reduction in the accuracy of standard secondary structure prediction methods for edge strands, in comparison with central strands.     

Patterns of nuclease protection during strand exchange. recA protein forms heteroduplex DNA by binding to strands of the same polarity

recA protein, in the presence of ATP, polymerizes on single-stranded DNA (plus strand) to form a presynaptic nucleoprotein filament that pairs with linear duplex DNA and actively displaces the plus strand from the recipient molecule in a polarized fashion to form a new heteroduplex molecule. The interaction between recA protein and DNA during strand exchange was studied by labeling different strands and probing the intermediate with pancreatic deoxyribonuclease I (DNase I) or restriction endonuclease. The incoming single strand was resistant to DNase I in the original nucleoprotein filament and remained resistant even after extensive strand exchange had occurred. Both strands of the parental duplex molecule were sensitive to DNase I in the absence of joint molecule formation; but as strand exchange progressed following homologous pairing, increasing stretches of the parental plus strand became resistant, whereas the complementary parental minus strand remained sensitive to DNase I throughout the reaction. Except for a region of 50-100 base pairs at the end of the newly formed heteroduplex DNA where strand exchange was initiated, the rest of the heteroduplex region was resistant to cleavage by restriction endonucleases. The data suggest that recA protein promotes strand exchange by binding both the incoming and outgoing strands of the same polarity, whereas the complementary strand, which must switch pairing partners, is unhindered by direct contact with the protein.     

Hydrogen bonding in globular proteins.

A global census of the hydrogen bonds in 42 X-ray-elucidated proteins was taken and the following demographic trends identified: (1) Most hydrogen bonds are local, i.e. between partners that are close in sequence, the primary exception being hydrogen-bonded ion pairs. (2) Most hydrogen bonds are between backbone atoms in the protein, an average of 68%. (3) All proteins studied have extensive hydrogen-bonded secondary structure, an average of 82%. (4) Almost all backbone hydrogen bonds are within single elements of secondary structure. An approximate rule of thirds applies: slightly more than one-third (37%) form i----i--3 hydrogen bonds, almost one-third (32%) form i----i--4 hydrogen bonds, and slightly less than one-third (26%) reside in paired strands of beta-sheet. The remaining 5% are not wholly within an individual helix, turn or sheet. (5) Side-chain to backbone hydrogen bonds are clustered at helix-capping positions. (6) An extensive network of hydrogen bonds is present in helices. (7) To a close approximation, the total number of hydrogen bonds is a simple function of a protein's helix and sheet content. (8) A unique quantity, termed the reduced number of hydrogen bonds, is defined as the maximum number of hydrogen bonds possible when every donor:acceptor pair is constrained to be 1:1. This quantity scales linearly with chain length, with 0.71 reduced hydrogen bond per residue. Implications of these results for pathways of protein folding are discussed.     

RecA-DNA filament topology: the overlooked alternative of an unconventional syn-syn duplex intermediate

The helical filaments of RecA protein mediate strand exchange for homologous recombination, but the paths of the interacting DNAs have yet to be determined. Although this interaction is commonly limited to three strands, it is reasoned here that the intrinsic symmetry relationships of quadruplex topology are superior in explaining a range of observations. In particular, this topology suggests the potential of post-exchange base pairing in the unorthodox configuration of syn-syn glycosidic bonds between the nucleotide bases and the pentose rings in the sugar-phosphate backbone, which would transiently be stabilized by the external scaffolding of the RecA protein filament.     

Structure and folding of a rare, natural kink turn in RNA with an A*A pair at the 2b*2n position

The kink turn (k-turn) is a frequently occurring motif, comprising a bulge followed by G•A and A•G pairs that introduces a sharp axial bend in duplex RNA. Natural k-turn sequences exhibit significant departures from the consensus, including the A•G pairs that form critical interactions stabilizing the core of the structure. Kt-23 found in the small ribosomal subunit differs from the consensus in many organisms, particularly in the second A•G pair distal to the bulge (2b•2n). Analysis of many Kt-23 sequences shows that the frequency of occurrence at the 2n position (i.e., on the nonbulged strand, normally G in standard k-turns) is U>C>G>A. Less than 1% of sequences have A at the 2n position, but one such example occurs in Thelohania solenopsae Kt-23. This sequence folds only weakly in the presence of Mg²⁺ ions but is induced to fold normally by the binding of L7Ae protein. Introduction of this sequence into the SAM-I riboswitch resulted in normal binding of SAM ligand, indicating that tertiary RNA contacts have resulted in k-turn folding. X-ray crystallography shows that the T. solenopsae Kt-23 adopts a standard k-turn geometry, making the key, conserved hydrogen bonds in the core and orienting the 1n (of the bulge-proximal A•G pair) and 2b adenine nucleobases in position facing the opposing minor groove. The 2b and 2n adenine nucleobases are not directly hydrogen bonded, but each makes hydrogen bonds to their opposing strands.     

Protein topology prediction through constraint-based search and the evaluation of topological folding rules.

An algorithm for predicting protein alpha/beta-sheet topologies from secondary structure and topological folding rules (constraints) has been developed and implemented in Prolog. This algorithm (CBS1) is based on constraint satisfaction and employs forward pruned breadth-first search and rotational invariance. CBS1 showed a 37-fold increase in efficiency over an exhaustive generate and test algorithm giving the same solution for a typical sheet of five strands whose topology was predicted from secondary structure with four topological folding constraints. Prolog specifications of a range of putative protein folding rules were then used to (i) replicate published protein topology predictions and (ii) validate these rules against known protein structures of nucleotide-binding domains. This demonstrated that (i) manual techniques for topology prediction can lead to non-exhaustive search and (ii) most of these protein folding principles were violated by specific proteins. Various extensions to the algorithm are discussed.     

A code governing specific binding of regulatory proteins to DNA and structure of stereospecific sites of regulatory proteins]

A model is proposed for the structure of stereospecific sites in regulatory proteins. On its basis a possible code is suggested that governs the binding of regulatory proteins at specific control sites on DNA. Stereospecific sites of regulatory proteins are assumed to contain pairs of antiparallel polypeptide chain segments which form a right-hand twisted antiparallel beta-sheet, with single-stranded regions at the ends of the beta-structure. The model predicts that binding reaction between a regulatory protein and double-helical DNA is a cooperative phenomenon and is accompanied by significant structural alteration at the stereospecific site of the protein. Half of hydrogen bonds normally existing in beta-structure are broken upon complex formation with DNA and a new set of hydrogen bonds is formed between polypeptide amide groups and DNA base pairs. In a stereospecific site, one chain (t-chain) is attached through hydrogen bonds to the carbonyl oxygens of pyramides and N3 adenines lying in one DNA strand, while the second polypeptide chain (g chain) is hydrogen bonded to the 2-amino groups of guanine residues lying in the opposite DNA strand. The amide groups serve as specific reaction sites being hydrogen bond acceptors in g-chain and hydrogen bond donors in t-chain. The single-stranded portions of t- and g-chains lying in neighbouring subunits of regulatory protein interact with each other forming deformed beta-sheets. The recognition of regulatory sequences by proteins is based on the structural complementarity between stereospecific sites of regulatory proteins and base pairs sequences at the control sites. An essential feature of these sequences is the asymmetrical distribution of guanine residues between the two DNA strands. The code predicts that there are six fundamental amino acid residues (serine, threonine, asparagine, histidine, glutamine and cysteine) whose sequence in stereospecific site determines the base pair sequence to which a given regulatory protein would bind preferentially. The code states a correspondence between four amino acid residues at the stereospecific site of regulatory protein with the two residues being in t- and g-segments, respectively, and AT(GC) base pair at the control site. It is thus possible to determine which amino acid residues in the repressor and which base pairs in the operator DNA are involved in specific interactions with each other, as exemplified by lac repressor binding to lac operator.     

Putative three-stranded DNA pairing intermediate in recA protein-mediated DNA strand exchange: no role for guanine N-7.

As an early step in DNA strand exchange reactions, the recA protein aligns homologous sequences within two DNA molecules to form a putative triple-stranded intermediate. In virtually all models for three-stranded DNA proposed to date, hydrogen bonds involving the N-7 position of guanine have played a prominent structural role. To determine whether the N-7 position of guanine is required for triple helix and heteroduplex formation in the recA protein-mediated DNA pairing reaction, guanine was completely replaced by the base analog 7-deazaguanine in both strands of the duplex DNA substrate using polymerase chain reaction. This modified double-strand DNA was reacted with unmodified single-strand DNA in vitro. The 7-deazaguanine-substituted DNA functioned as well as the unsubstituted DNA in recA protein-mediated DNA three-strand exchange reactions. Strand exchange reactions involving four strands also proceeded normally when three of the four strands contained 7-deazaguanine rather than guanine. In fact, the rate of strand exchange improved somewhat when the modified DNA substrates were used. This indicates either that the N-7 position of guanine is not essential for the formation of the putative triple-stranded DNA pairing intermediate, or that a three-stranded (or four-stranded) structure is not an obligate intermediate in recA protein-mediated DNA strand exchange.     

A novel palindromic triple-stranded structure formed by homopyrimidine dodecamer d-CTTCTCCTCTTC and homopurine hexamer d-GAAGAG.

We have carried out NMR and molecular mechanics studies on a complex formed when a palindromic homopyrimidine dodecamer (d-CTTCTCCTCTTC) and a homopurine hexamer (d-GAAGAG) are mixed in 1:1 molar ratio in aqueous solutions. Such studies unequivocally establish that two strands of each oligomer combine to form a triple-stranded DNA structure with a palindromic symmetry and with six T.A:T and six C+. G:C hydrogen-bonded base triads. The two purine strands are placed head to head, with their 3' ends facing each other in the center of the structure. One-half of each pyrimidine strand contains protonated and the other half contains non-protonated cytosines. The two half segments containing protonated cytosines are hydrogen bonded to each of the two purine hexamers through Hoogsteen T.A and C+.G base pairing. The segments containing non-protonated cytosines are involved in Watson-Crick (A:T and G:C) base pairing. This leads to a palindromic triplex with a C2-dyad symmetry with respect to the center of the structure. The complex is less stable at neutral pH, but the cytosines involved in Hoogsteen base pairing remain protonated even under these conditions. Molecular mechanics calculations using NMR constraints have provided a detailed three-dimensional structure of the complex. The entire stretches of purine, and the pyrimidine nucleotides have a conformation close to B-DNA.     

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel approach for alpha-helical topology prediction in globular proteins: generation of interhelical restraints

The protein folding problem represents one of the most challenging problems in computational biology. Distance constraints and topology predictions can be highly useful for the folding problem in reducing the conformational space that must be searched by deterministic algorithms to find a protein structure of minimum conformational energy. We present a novel optimization framework for predicting topological contacts and generating interhelical distance restraints between hydrophobic residues in alpha-helical globular proteins. It should be emphasized that since the model does not make assumptions about the form of the helices, it is applicable to all alpha-helical proteins, including helices with kinks and irregular helices. This model aims at enhancing the ASTRO-FOLD protein folding approach of Klepeis and Floudas (Journal of Computational Chemistry 2003;24:191-208), which finds the structure of global minimum conformational energy via a constrained nonlinear optimization problem. The proposed topology prediction model was evaluated on 26 alpha-helical proteins ranging from 2 to 8 helices and 35 to 159 residues, and the best identified average interhelical distances corresponding to the predicted contacts fell below 11 A in all 26 of these systems. Given the positive results of applying the model to several protein systems, the importance of interhelical hydrophobic-to-hydrophobic contacts in determining the folding of alpha-helical globular proteins is highlighted.     

The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain

Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.     

Interplay between hydrophobic cluster and loop propensity in beta-hairpin formation

Autonomously folding beta-hairpins have recently emerged as powerful tools for elucidating the origins of antiparallel beta-sheet folding preferences. Analysis of such model systems has suggested four potential sources of beta-sheet stability: (1) the conformational propensity of the loop segment that connects adjacent strands; (2) favorable contacts between side-chains on adjacent strands; (3) interstrand hydrogen bonds; and (4) the intrinsic beta-sheet propensities of the strand residues. We describe the design and analysis of a series of isomeric 20 residue peptides in which factors (1)-(4) are identical. Differences in beta-hairpin formation within this series demonstrate that these four factors, individually, are not sufficient to explain beta-sheet stability. In agreement with the prediction of a simple statistical mechanical model for beta-hairpin formation, our results show that the separation between the loop segment and an interstrand cluster of hydrophobic side-chains strongly influences beta-hairpin size and stability, with a smaller separation leading to greater stability.     

Global optimisation by replica exchange with scaled hybrid Hamiltonians

A global optimisation scheme based on replica-exchange molecular dynamics simulation with scaled hybrid Hamiltonians is presented and applied to fold trpzip2 peptide from extended structures with explicit water model using only eight replicas. The algorithm is shown to be capable of reproducibly optimising the peptide to structures of root mean squared deviations less than 2.5 Å with respect to all heavy atoms of NMR models. Moreover, the large amount of structural data sampled in the optimisation process enables us to provide a possible folding mechanism. The transition state ensemble is characterised by a largely formed turn and a compact packing of tryptophan 2, 4 and 9. The tryptophan 2/11 pair is found to form at a very late stage of the folding process. The first (closest to the turn) and the fourth native backbone hydrogen bonds form earlier and a picture of strict zipping up of hydrogen bonds is not observed. It is demonstrated in the present study that this global optimisation method which integrates structure prediction with approximated conformational samplings may be of help to understand the folding puzzle.     

Contributions of residue pairing to beta-sheet formation: conservation and covariation of amino acid residue pairs on antiparallel beta-strands

In an effort to better understand beta-sheet assembly, we have investigated the evolutionary behavior of neighboring residues on adjacent antiparallel beta-strands. Residue pairs were classified according to solvent exposure as well as by whether their backbone NH and C==O groups are hydrogen bonded. The conservation and covariation of 19,241 pairs in 219 sequence alignments was analyzed. Buried pairs were found to be the most conserved, while stronger covariation was detected in the solvent-exposed pairs. However, residues on neighboring strands showed a degree of conservation and covariation similar to that of well-separated residues on the same strand, suggesting that evolutionary pressure to maintain complementarity between pairs on neighboring strands is weak. Moreover, in spite of the preference of certain amino acid pairs to occupy neighboring positions on adjacent strands, such favored pairs are neither more strongly mutually conserved nor covary more strongly than pairs of the same type in non-interacting positions. Although the beta-sheet pairs did not show outstanding evolutionary coupling, in many protein families significant conservation and covariation patterns were detected for some of the residue pairs. Overall, the weak evolutionary conservation and covariation of the beta-sheet pairs indicates that sheet structure is unlikely to be dictated by specific side-chain interactions.     

Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products.     

Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.