Abstract: High-accuracy ab initio folding has remained an elusive objective despite decades of effort. To explore the folding landscape of villin headpiece subdomain HP35, we conducted two sets of replica exchange molecular dynamics for 200 ns each and three sets of conventional microsecond-long molecular dynamics simulations, using AMBER FF03 force field and a generalized-Born solvation model. The protein folded consistently to the native state; the lowest C(alpha)-rmsd from the x-ray structure was 0.46 A, and the C(alpha)- rmsd of the center of the most populated cluster was 1.78 A at 300 K. ab initio simulations have previously not reached this level. The folding landscape of HP35 can be partitioned into the native, denatured, and two intermediate-state regions. The native state is separated from the major folding intermediate state by a small barrier, whereas a large barrier exists between the major folding intermediate and the denatured states. The melting temperature T(m) = 339 K extracted from the heat-capacity profile was in close agreement with the experimentally derived T(m) = 342 K. A comprehensive picture of the kinetics and thermodynamics of HP35 folding emerges when the results from replica exchange and conventional molecular dynamics simulations are combined.

Abstract: Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations. Even though some unfolded and partially folded proteins are now known to possess biological function or to be involved in aggregation phenomena associated with disease states, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR pulse-labelling experiments that involve rapid in situ protein refolding. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.

Abstract: DExD/H-box proteins are ubiquitously involved in RNA-mediated processes and use ATP to accelerate conformational changes in RNA. However, their mechanisms of action, and what determines which RNA species are targeted, are not well understood. Here we show that the DExD/H-box protein CYT-19, a general RNA chaperone, mediates ATP-dependent unfolding of both the native conformation and a long-lived misfolded conformation of a group I catalytic RNA with efficiencies that depend on the stabilities of the RNA species but not on specific structural features. CYT-19 then allows the RNA to refold, changing the distribution from equilibrium to kinetic control. Because misfolding is favoured kinetically, conditions that allow unfolding of the native RNA yield large increases in the population of misfolded species. Our results suggest that DExD/H-box proteins act with sufficient breadth and efficiency to allow structured RNAs to populate a wider range of conformations than would be present at equilibrium. Thus, RNAs may face selective pressure to stabilize their active conformations relative to inactive ones to avoid significant redistribution by DExD/H-box proteins. Conversely, RNAs whose functions depend on forming multiple conformations may rely on DExD/H-box proteins to increase the populations of less stable conformations, thereby increasing their overall efficiencies.

Abstract: The electrospray ionization (ESI) charge state distribution of proteins is highly sensitive to the protein structure in solution. Unfolded conformations generally form higher charge states than tightly folded structures. The current study employs a minimalist molecular dynamics model for simulating the final stages of the ESI process in order to gain insights into the physical reasons underlying this empirical relationship. The protein is described as a string of 27 beads ("residues"), 9 of which are negatively charged and represent possible protonation sites. The unfolded state of this bead string is a random coil, whereas the native conformation adopts a compact fold. The ESI process is simulated by placing the protein inside a solvent droplet with a 2.5 nm radius consisting of 1600 Lennard-Jones particles. In addition, the droplet contains 14 protons which are modeled as highly mobile point charges. Disintegration of the droplet rapidly releases the protein into the gas phase, resulting in average charge states of 4.8+ and 7.4+ for the folded and unfolded conformation, respectively. The protonation probabilities of individual residues in the folded state reveal a characteristic pattern, with values ranging from 0.2 to 0.8. In contrast, the protonation probabilities of the unfolded protein are more uniform and cover the range from 0.8 to 1.0. The origin of these differences can be traced back to a combination of steric and electrostatic effects. Residues exhibiting a small accessible surface area are less likely to capture a proton, an effect that is exacerbated by partial electrostatic shielding from nearby positive residues. Conversely, sites that are sterically exposed are associated with electrostatic funnels that greatly increase the likelihood of protonation. Unfolding enhances the steric and electrostatic exposure of protonation sites, thereby causing the protein to capture a greater number of protons during the droplet disintegration process.

Abstract: The importance of disordered protein states in biology is gaining recognition, and can be attributed in part to the participation of unfolded and partially folded states of globular proteins in normal and abnormal biological functions, such as protein translation, protein translocation, protein degradation, protein assembly, and protein aggregation (1-5). There is also a growing awareness that a significant fraction of gene products from various genomes, including the human genome, fall into a category that includes low complexity, low globularity, or intrinsically unstructured proteins (6-9). Unlike native states of globular proteins, disordered protein states, by definition, do not adopt a fixed structure that can be determined using classical high-resolution methods. Nevertheless, there has long been evidence that many disordered states contain detectable and significant residual or nascent structure (10-16). This structure has been found to be important for nucleating local structure, as well as mediating long range contacts upon either intramolecular folding to the native state (17-21) or intermolecular folding with specific binding partners (22-24), and is also predicted to influence intermolecular folding into structured aggregates (25,26). The primary tool for the characterization of such structure is high-resolution solution state nuclear magnetic resonance (NMR) spectroscopy. Advances in NMR instrumentation and methods have greatly facilitated this task and in principle can now be accomplished by those without extensive prior experience in NMR spectroscopy. This chapter describes how this can be accomplished.

Abstract: Prolonged heating of holo bovine alpha-lactalbumin (BLA) at 80 degrees C in pH 7 phosphate buffer in the absence of a thiol initiator improves the surface activity of the protein at the air:water interface, as determined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzed on cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increases with the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLA are present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMR characterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heated for 120 min showed that it consisted of a mixture of refolded native protein and partially folded protein and that the partially folded protein species had spectral characteristics similar to those of the pH 2 molten globule state of the protein. Circular dichroism spectroscopy showed that the non-native species had approximately 40% of the alpha-helical content of the native state, but lacked persistent tertiary interactions. Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolated by high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containing the non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largely responsible for the pronounced improvement in surface activity of the protein on heating.

Abstract: The venoms of marine predatory cone snails contain cocktails of pharmacologically active peptides. These Conus peptides are broadly classified into two groups: (a) possessing a single or no disulfide bridge and (b) having multiple disulfide bridges. The latter group forms the major components of the Conus venom (1). These peptides, collectively named as conotoxins, form compact and well-ordered three-dimensional (3D) structures through the formation of two or more disulfide bridges among the short sequence length of 10–30 amino acid residues (1–3). So far, an extensive listing of conotoxins have been purified and characterized. Depending on the characteristic disulfide linkage patterns seen in the mature peptides as well as the highly conserved signal sequences of the precursor molecules, these toxins are classified into the A-, M-, O-, P-, S-, and T-superfamilies (2). On the basis of their cysteine framework, the first three superfamilies are subdivided into various families. Members of each family of conotoxins possess identical cysteine framework as well as remarkable molecular specificity to recognize a precise subclass of ion channels/receptors (1–3). α-Conotoxins, which belong to the A-superfamily, are one of the most intensely studied groups of conotoxins. Functionally, they are competitive inhibitors of either “muscle-type” or “neuronal-type” nicotinic acetylcholine receptors (2). Structurally, α-conotoxins possess 11–19 amino acid residues including four highly conserved cysteine residues and fold into the native globular conformation, with a specific cysteine pairing (C1–3, C2–4). In contrast, the two non-native forms (which suffer at least a 10-fold reduction in biological activity) are not found in nature (4). To determine the contribution of the side chains toward the formation of the globular conformation, Zhang and Snyder studied the effect of alanine substitution on several conserved residues in the 3/5 α-conotoxin GI (5). However, substitution with alanine, either singly or in combination, failed to result in the total conformational switch from the native conformation to either of the two non-native forms. Substitutions included proline and glycine that are likely to induce a kink or flexibility in the sequence and often dictate folding patterns (6, 7). The influence of the number of residues within the intercysteine loops was also examined by changing the position of the third cysteine residue in the peptide sequence. The authors concluded that for α-conotoxin sequences with intercysteine loops comprising less than five residues number rather than type of residues was more critical for achieving a globular conformation; loops with an even number of residues appeared to favor the globular conformation (5). However, this rule does not appear applicable to the different subclasses of α-conotoxin, especially in the prediction of the globular versus the ribbon conformations (8). Recently, several groups discovered a new family of conotoxins: χ-conotoxins (or λ-conotoxin) (9–11). This novel class of conotoxins possesses the conserved arrangement of quadruple cysteines as α-conotoxins. However, their native cysteine pairing is unique as they exhibit a ribbon (C1–4, C2–3) conformation instead of the usual globular structure seen in α-conotoxins. The mechanism of neurotoxicity induced by the χ/λ-conotoxins is also distinct from that of α-conotoxins (10). As in the case of α-conotoxins, the native cysteine pairing and consequent conformation have also been shown to be important for the biological activity of χ/λ-conotoxins: the non-native globular isoform of χ/λ-conotoxin CMrVIA displays a lower potency of three orders in magnitude in the induction of seizures in mice (9). These findings underscore the crucial role of disulfide linkages and conformation for the biological potencies of these peptides. However, the structural features governing the disulfide pattern and attendant conformational differences are still unclear. The discovery of χ/λ-conotoxins lends insights into the structural features that are critical for the differing folding patterns of these two families of Conus peptides. To delineate structural determinants responsible for the shift in folding tendency, we systematically analyzed the structural differences between α-conotoxins and χ/λ-conotoxins. Herein we describe the contribution of the proline residue in the first intercysteine loop in affecting the folding pattern in representative members of these families of conotoxins. By examining further variants, we also confirm our earlier finding on the effect of C-terminal amidation on the folding pattern of ImI α-conotoxin (12). By using synthetic congeners of ImI conotoxin and parallel NMR studies of the various folded forms, we show that these structural determinants, individually and in combination, shift the pattern toward the globular conformation. These structural changes of various ImI conotoxin analogues are correlated with their ability to bind to Aplysia and Bulinus acetylcholine binding proteins (abbreviated as A-AChBP and B-AChBP, respectively), the protein family of which structures of α-conotoxins at their binding sites have been reported (13, 14). In turn, by substituting proline into the first intercysteine loop of CMrVIA χ/λ-conotoxin, we show that the native ribbon conformation of CMrVIA conotoxin reciprocally folds into its non-native globular form.

Abstract: The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N --> I --> U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (DeltaGH2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 +/- 0.51 kcal mol-1; however, the DeltaGH2O for the intermediate species at pH 4 is 2.01 +/- 0.47 kcal mol-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 +/- 0.15 and 1.34 +/- 0.17 kcal mol-1 M-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pKa values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pKa values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D 1H-15N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-l-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.

Abstract: High-resolution protein separation procedures are an important prerequisite for proteome analyses. Classically, protein separations are based on 2D IEF/SDS-PAGE. Unfortunately, this technique only poorly recovers hydrophobic proteins, and it is not compatible with analyses of proteins in native state. Blue-native PAGE represents a powerful alternative. It is based on the careful integration of negative charge into proteins and protein complexes by the anionic wool dye Coomassie blue, and it allows protein analyses under native ("blue-native") conditions. Upon combination with SDS-PAGE for a second gel dimension, protein complexes separated by the blue-native dimension are dissected into their subunits, which form vertical rows of spots on the resulting 2D gels. 2D blue-native/SDS-PAGE ideally complements 2D IEF/SDS-PAGE in proteomics.

Abstract: Adoption of a specific three-dimensional structure for RNA is a complex process that typically involves the formation and accumulation of intermediates. Most fundamentally, this behavior arises because local RNA structure can form rapidly and can be stable in the absence of enforcing long-range or global structure, allowing partially structured intermediates to form and accumulate. The stability of RNA structure may lead, in general, to a hierarchy in its folding process, with global tertiary structure forming primarily from pre-formed units of secondary and local tertiary structure (5). This folding behavior contrasts with that of many proteins, whose local elements of secondary and tertiary structure are unstable in the absence of the global fold of a subunit or domain and are therefore formed in concert (6, 7). This hierarchy may facilitate the folding of RNAs, as stable structure that forms early can provide a scaffold upon which additional structure is built. However, hierarchical folding also introduces the possibility that incorrect structure will form and be stable, generating misfolded or ‘kinetically trapped’ intermediates, which require partial or complete unfolding to continue folding productively. Indeed, RNA misfolding has been recognized since early studies of tRNA (8, 9) and has more recently been shown to be a near-ubiquitous feature of folding for large, multi-domain RNAs (10-16). A wealth of information on folding steps and intermediates has been obtained from studies of the Tetrahymena group I ribozyme. The ribozyme includes a core and several peripheral elements that surround the core. Several lines of evidence have indicated that one of the peripheral elements, termed P5abc, plays a vital role in folding. Time-resolved oligonucleotide hybridization and footprinting studies showed that tertiary folding begins with P5abc, followed closely by the rest of the P4-P6 domain (2, 3, 17, 18) (Fig. 1). The rest of the ribozyme then acquires stable structure through a series of intermediates (2, 3, 18), while P5abc and the entire P4-P6 domain retain structure in each subsequent intermediate. In addition to being the first subdomain to form, P5abc is the most stable part of the ribozyme, remaining ordered at the lowest Mg2+ concentration and the highest temperature (19, 20). Consistent with the view of P5abc as a critical part of the structure, its deletion gives a ribozyme (EΔP5abc) that is substantially destabilized and has greatly diminished catalytic activity (4, 21), defects that are rescued by addition of P5abc as a separate molecule (4, 22). Because of its stability and rapid formation of tertiary structure, P5abc was proposed to facilitate folding by nucleating formation of the P4-P6 domain, which would then provide the scaffold for the entire ribozyme (3, 23). Figure 1 Tertiary folding of the Tetrahymena ribozyme. Double-stranded regions that do not have stable tertiary structure are shown as gray cylinders, and regions that have formed tertiary structure are colored as follows. The core domains, P4-P6 and P3-P8, are ... On the other hand, studies of folding kinetics called into question the productive role of early P5abc folding. Several mutations that weaken native structure within P5abc increase the rate of stable tertiary structure formation within the P3-P8 domain, suggesting that native structure formed early within P5abc is transiently disrupted at a later stage of folding (14, 24, 25), and similar mutations accelerate refolding of a long-lived misfolded intermediate that is formed by most of the ribozyme population during folding [(26); see also (15, 27, 28)]. Further, the small fraction of the ribozyme that avoids the misfolded conformation folds only slightly more slowly upon deletion of P5abc (15). These studies indicated that under the conditions of these experiments (10 mM Mg2+ and <20 mM Na+, ‘low salt’ conditions), the early formation of P5abc provides no overall benefit and even appears to slow folding steps involving the ribozyme core. However, it remained possible that P5abc accelerated early folding steps, but the beneficial effect was not detected because it was obscured by later steps that were unaffected or slowed by P5abc. More recently, it has been shown that mutation of a tertiary contact within P5abc or addition of the denaturant urea to the wild-type ribozyme accelerates even early folding steps, most simply suggesting that structure within P5abc is detrimental even early in folding (29). To more fully understand the role of P5abc structure in folding, here we use footprinting and activity assays to systematically examine early and late folding steps, comparing folding of the wild-type ribozyme and the EΔP5abc variant under ‘low salt’ solution conditions. Strikingly, we find that deletion of P5abc accelerates even the early folding steps, suggesting that, under these conditions, most or all of the partially structured intermediates that accumulate are kinetically trapped. Further, although P5abc has little effect on the rate constant for native state formation along the sparsely populated pathway that avoids the long-lived misfolded conformation, it dramatically slows refolding of the heavily populated misfolded intermediate. Finally, we build on previous studies by showing that P5abc stabilizes the native state relative to partially structured and unstructured intermediates, supporting previous results in providing a rationale for its presence in the natural RNA even if the deleterious effects observed here are also present during folding in vivo.

Abstract: Much knowledge of protein folding can be derived from the examination of the nature and size of solvent-exposed surfaces along conformational transitions. We exploit here a general photochemical modification with methylene carbene of the accessible surface area (ASA) of the polypeptide chain. Labeling of Bacillus licheniformis β-lactamase (BL-βL) with 1 mM 3H-diazirine yielded 8.3 × 10-3 mol CH2/mol protein, in agreement with the prediction for an unspecific surface labeling phenomenon. The unfolded state U in 7 M urea was labeled 60% more than the native state N. This result lies well below the increment of ASA expected from theoretical estimates and points to the presence of residual organization in state U and/or of cavities or crevices favoring the partition of the reagent in state N. A partially folded state I was demonstrated from two sequential transitions occurring at 1.5−3.0 M and 3.5−6.5 M urea. This technique shows a close correlation with optical probes most sensitive to changes in tertiary st...

Abstract: Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native beta-actin, but is unable to guide beta-actin toward the native state. In this study we identify conformational rearrangements in beta-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of beta-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled beta-actin was characterized, and the structural rearrangement was compared to the case of the beta-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarstrom, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the beta-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound beta-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the beta-actin molecule. It can be concluded that the specific and directed rearrangement of the beta-actin structure, seen in the natural beta-actin-TRiC system, is vital for guiding beta-actin to the native state.