Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy.
ABSTRACT: To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.
Project description:Left-handed polyproline II (PPII) helices commonly occur in globular proteins in segments of 4-8 residues. This paper analyzes the structural conservation of PPII-helices in 3 protein families: serine proteinases, aspartic proteinases, and immunoglobulin constant domains. Calculations of the number of conserved segments based on structural alignment of homologous molecules yielded similar results for the PPII-helices, the alpha-helices, and the beta-strands. The PPII-helices are consistently conserved at the level of 100-80% in the proteins with sequence identity above 20% and RMS deviation of structure alignments below 3.0 A. The most structurally important PPII segments are conserved below this level of sequence identity. These results suggest that the PPII-helices, in addition to the other 2 secondary structure classes, should be identified as part of structurally conserved regions in proteins. This is supported by similar values for the local RMS deviations of the aligned segments for the structural classes of PPII-helices, alpha-helices, and beta-strands. The PPII-helices are shown to participate in supersecondary elements such as PPII-helix/alpha-helix. The conservation of PPII-helices depends on the conservation of a supersecondary element as a whole. PPII-helices also form links, possibly flexible, in the interdomain regions. The role of the PPII-helices in model building by homology is 2-fold; they serve as additional conserved elements in the structure allowing improvement of the accuracy of a model and provide correct chain geometry for modeling of the segments equivalenced to them in a target sequence. The improvement in model building is demonstrated in 2 test studies.
Project description:Local structures in denatured proteins may be important in guiding a polypeptide chain during the folding and misfolding processes. Existence of local structures in chemically denatured proteins is a highly controversial issue. NMR parameters [coupling constants (3) J(H(alpha),H(N)) and chemical shifts] of chemically denatured proteins in general deviate little from their values in small peptides. These peptides were presumed to be completely unstructured; therefore, it was considered that chemically denatured proteins are random coils. But recent experimental studies show that small peptides adopt relatively stable structures in aqueous solutions. Small deviations of the NMR parameters from their values in small peptides may thus actually indicate the existence of local structures in chemically denatured proteins. Using NMR data and theoretical predictions we show here that fluctuating beta-strands exist in urea-denatured ubiquitin (8 M urea at pH 2). Residues in such beta-strands populate more frequently the left side of the broad beta region of -psi space. Urea-denatured ubiquitin contains no detectable beta-sheet secondary structures; nevertheless, the fluctuating beta-strands in urea-denatured ubiquitin coincide to the beta-strands in the native state. Formation of beta-strands is in accord with the electrostatic screening model of unfolded proteins. The free energy of a residue in an unfolded protein is in this model determined by the local backbone electrostatics and its screening by backbone solvation. These energy terms introduce strong electrostatic coupling between neighboring residues, which causes cooperative formation of beta-strands in denatured proteins. We propose that fluctuating beta-strands in denatured proteins may serve as initiation sites to form fibrils.
Project description:Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.
Project description:Polyproline II (PPII) is reported to be a dominant conformation in the unfolded state of peptides, even when no prolines are present in the sequence. Here we use isothermal titration calorimetry (ITC) to investigate the PPII bias in the unfolded state by studying the binding of the SH3 domain of SEM-5 to variants of its putative PPII peptide ligand, Sos. The experimental system is unique in that it provides direct access to the conformational entropy change of the substituted amino acids. Results indicate that the denatured ensemble can be characterized by at least two thermodynamically distinct states, the PPII conformation and an unfolded state conforming to the previously held idea of the denatured state as a random collection of conformations determined largely by hard-sphere collision. The probability of the PPII conformation in the denatured states for Ala and Gly were found to be significant, approximately 30% and approximately 10%, respectively, resulting in a dramatic reduction in the conformational entropy of folding.
Project description:Conformational equilibria in the protein denatured state have key roles regulating folding, stability, and function. The extent of conformational bias in the protein denatured state under folding conditions, however, has thus far proven elusive to quantify, particularly with regard to its sequence dependence and energetic character. To better understand the structural preferences of the denatured state, we analyzed both the sequence dependence to the mean hydrodynamic size of disordered proteins in water and the impact of heat on the coil dimensions, showing that the sequence dependence and thermodynamic energies associated with intrinsic biases for the ? and polyproline II (PPII) backbone conformations can be obtained. Experiments that evaluate how the hydrodynamic size changes with compositional changes in the protein reveal amino acid specific preferences for PPII that are in good quantitative agreement with calorimetry-measured values from unfolded peptides and those inferred by survey of the protein coil library. At temperatures above 25 °C, the denatured state follows the predictions of a PPII-dominant ensemble. Heat effects on coil hydrodynamic size indicate the ? bias is comparable to the PPII bias at cold temperatures. Though historically thought to give poor resolution to structural details, the hydrodynamic size of the unfolded state is found to be an effective reporter on the extent of the biases for the ? and PPII backbone conformations.
Project description:Solid-state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid-state (13)C NMR spectra of samples prepared with uniform (13)C labeling of selected residues show relatively sharp cross-peaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most cross-peaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid-state NMR techniques that provide independent constraints on backbone varphi and psi torsion angles in samples with sequential pairs of carbonyl (13)C labels. Without GdnHCl, the combined data are well fit by alpha-helical conformations. At [GdnHCl]=4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of alpha-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl]=7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid-state NMR.
Project description:It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59-heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~27 ?s of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency>90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.
Project description:Raman optical activity (ROA) spectra of proteins hold a lot of information about their structure in solution. To create a better understanding of the ROA spectra of, among others, the intrinsically disordered proteins (IDPs), involved in neurodegenerative diseases, the effect of conformational disorder and dynamics on the ROA spectra was studied. Density functional theory (DFT) calculations of small ensembles of model peptides with increasing disorder show that the ROA patterns of ?-helical and polyproline II (PPII) structure reflect the average backbone angles in the ensemble. The amide III region in the ROA spectra of the ?-helical peptides is shown to retain its typical -/+/+ pattern, while the amide III region of PPII secondary structure diminishes in intensity with increasing structural disorder. The results show that the ROA spectra of IDPs hence more likely stem from short stretches of well-defined PPII helices rather than a very flexible chain. Further DFT calculations support that mixing of PPII with helical secondary structure is consistent with experimental spectra of IDPs, while mixing with ?-strand results in spectral patterns that are not observed experimentally. The detailed information obtained from these results contributes to a better understanding of the spectrum-structure relation.
Project description:To understand the structural basis of molecular elasticity and protein interaction of the elastic PEVK (Pro-Glu-Val-Lys) segment of the giant muscle protein titin, we carried out a detailed analysis of a representative PEVK module and a 16-module PEVK protein under various environmental conditions. Three conformational states, polyproline II (PPII) helix, beta-turn and unordered coil were identified by CD and NMR. These motifs interconvert without long-range co-operativity. As a general trend, the relative content of PPII increases with lower temperature and higher polarity, beta-turn increases with lower temperature and lower polarity, and unordered coil increases with higher temperature and higher polarity. NMR studies demonstrate that trans -proline residues are the predominant form at room temperature (22 degrees C), with little trans -to- cis isomerization below 35 degrees C. Ionic strength affects salt bridges between charged side chains, but not the backbone conformation. We conclude that titin PEVK conformation is malleable and responds to subtle environmental changes without co-operativity. This gradual conformational transition may represent a regulatory mechanism for fine-tuning protein interactions and elasticity.
Project description:Denaturation of Bordetella pertussis fimbrial preparations by guanidinium hydrochloride (GdnHCl) has been characterized using static light scattering, c.d., fluorescence and antibody recognition. The susceptibility of Fim2 + 3 (a mixed preparation of two fimbrial types) to GdnHCl was found to be highly dependent on pH; as the pH was increased from pH 7.2 to 10.5, the concentration of GdnHCl required to induce 50% denaturation was decreased. At pH 10.5, Fim2 + 3 was denatured by GdnHCl in a three-step pathway comprising: (1) formation of a pre-denaturational intermediate at less than 1.0 M-GdnHCl; (2) dissociation of the fimbrial polymer into subunits between 2 M- and 3.2 M-GdnHCl; and (3) subunit unfolding between 2.8 M- and 3.6 M-GdnHCl. A similar pathway was also found for the denaturation of the individual fimbrial types, Fim2 and Fim3, except that unfolding of either subunit commenced at a lower GdnHCl concentration (2.2 M) than that found for the mixture of fimbriae, Fim2 + 3. The second step in the denaturation pathway, dissociation into subunits, was partially reversible, but the renaturation and reassociation of fully unfolded subunits to form fimbriae-like structures was not achieved. These findings demonstrate that the GdnHCl denaturation of complex polymeric proteins is unlikely to follow a reversible two-state denaturation pathway, and support the involvement of a chaperone-like protein in the folding and assembly of the fimbriae in vivo. Measurement of the ability of anti-fimbrial monoclonal antibodies to recognize intermediates in the denaturation pathway enabled the identification of two types of epitope which were dependent on different aspects of fimbrial tertiary/quaternary structure.