Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR*

The susceptibility of the cellular prion protein (PrP^C) to convert to an alternative misfolded conformation (PrP^Sc), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23–231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a β-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4.     

未折叠蛋白质的普遍初始热力学亚稳态

探索和理解蛋白质折叠问题一直是分子生物学、结构生物学和生物物理学的终极挑战.未折叠的蛋白质应该存在一种普遍初始热力学亚稳态,否则无法解释蛋白质是如何在剧烈的热振动干扰下完成快速精确折叠的.本文通过分析水溶液环境和蛋白质折叠的相关性,揭示了一种由水分子屏蔽效应引起的未折叠蛋白质的普遍初始热力学亚稳态,该亚稳态的存在是水溶液环境中水分子的物理性质决定,并赋予未折叠蛋白质抵抗热扰动和避免错误折叠的能力.我们通过研究已发表的实验数据和建立分子模型,找到了该初始热力学亚稳态存在的相关证据,并推测了该亚稳态导致蛋白质精确折叠的相关物理学机制.     

Predicting the redox state and secondary structure of cysteine residues in proteins using NMR chemical shifts

Different programs and methods were employed to superimpose protein structures, using members of four very different protein families as test subjects, and the results of these efforts were compared. Algorithms based on human identification of key amino acid residues on which to base the superpositions were nearly always more successful than programs that used automated techniques to identify key residues. Among those programs automatically identifying key residues, MASS could not superimpose all members of some families, but was very efficient with other families. MODELLER, MultiProt, and STAMP had varying levels of success. A genetic algorithm program written for this project did not improve superpositions when results from neighbor-joining and pseudostar algorithms were used as its starting cases, but it always improved superpositions obained by MODELLER and STAMP. A program entitled PyMSS is presented that includes three superposition algorithms featuring human interaction.     

Vivaldi: Visualization and validation of biomacromolecular NMR structures from the PDB

We describe Vivaldi (VIsualization and VALidation DIsplay; http://pdbe.org/vivaldi ), a web‐based service for the analysis, visualization, and validation of NMR structures in the Protein Data Bank (PDB). Vivaldi provides access to model coordinates and several types of experimental NMR data using interactive visualization tools, augmented with structural annotations and model‐validation information. The service presents information about the modeled NMR ensemble, validation of experimental chemical shifts, residual dipolar couplings, distance and dihedral angle constraints, as well as validation scores based on empirical knowledge and databases. Vivaldi was designed for both expert NMR spectroscopists and casual non‐expert users who wish to obtain a better grasp of the information content and quality of NMR structures in the public archive. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Partially unfolded forms and non-two-state folding of a beta-sandwich: FHA domain from Arabidopsis receptor kinase-associated protein phosphatase

FHA domains adopt a beta-sandwich fold with 11 strands. The first evidence of partially unfolded forms of a beta-sandwich is derived from native-state hydrogen exchange (NHX) of the forkhead-associated (FHA) domain from kinase-associated protein phosphatase from Arabidopsis. The folding kinetics of this FHA domain indicate that EX2 behavior prevails at pH 6.3. In the chevron plot, rollover in the folding arm and bends in the unfolding arm suggest folding intermediates. NHX of this FHA domain suggests a core of six most stable beta-strands and two loops, characterized by rare global unfolding events. Flanking this stable core are beta-strands and recognition loops with less stability, termed subglobal motifs. These suggest partially unfolded forms (near-native intermediates) with two levels of stability. The spatial separation of the subglobal motifs on the flanks suggests possible parallelism in their folding as additional beta-strands align with the stable core of six strands. Intermediates may contribute to differences in stabilities and m-values suggested by NHX or kinetics relative to chemical denaturation. Residual structure in the unfolded regime is suggested by superprotection of beta-strand 6 and by GdmCl-dependence of adjustments in amide NMR spectra and residual optical signal. The global folding stability depends strongly on pH, with at least 3 kcal/mol more stability at pH 7.3 than at pH 6.3. This FHA domain is hypothesized to fold progressively with initial hydrophobic collapse of its stable six-stranded core followed by addition of less stable flanking beta-strands and ordering of recognition loops.     

Evolutionarily Conserved Proline Residues Impede the Misfolding of the Mouse Prion Protein by Destabilizing an Aggregation-competent Partially Unfolded Form

The misfolding of the prion protein has been linked to several neurodegenerative diseases. Despite extensive studies, the mechanism of the misfolding process remains poorly understood. The present study structurally delineates the role of the conserved proline residues present in the structured C-terminal domain of the mouse prion protein (moPrP) in the misfolding process. It is shown that mutation of these Pro residues to Ala leads to destabilization of the native (N) state, and also to rapid misfolding. Using hydrogen–deuterium exchange (HDX) studies coupled with mass spectrometry (MS), it has been shown that the N state of moPrP is in rapid equilibrium with a partially unfolded form (PUF2*) at pH 4. It has been shown that the Pro to Ala mutations make PUF2* energetically more accessible from the N state by stabilizing it relative to the unfolded (U) state. The apparent rate constant of misfolding is found to be linearly proportional to the extent to which PUF2* is populated in equilibrium with the N state, strongly indicating that misfolding commences from PUF2*. It has also been shown that the Pro residues restrict the boundary of the structural core of the misfolded oligomers. Overall, this study highlights how the conserved proline residues control misfolding of the prion protein by modulating the stability of the partially unfolded form from which misfolding commences.     

Estimation of protein secondary structure content directly from NMR spectra using an improved empirical correlation with averaged chemical shift

We have recently shown that the averaged chemical shift (ACS) of a nucleus in the protein backbone correlates well empirically to its secondary structure content (SSC). This allows the estimation of SSC directly from the NMR spectrum without the time intensive process of chemical shift assignment. Here, we present an empirical correlation that accounts both for contributions to the relevant protein and chemical shift databases made subsequent to the original analysis, and for missing or inconsistently referenced resonances. Our results affirm that this method provides a significant tool for initial structural prediction from NMR data prior to complete chemical shift assignment.     

Automated NMR determination of protein backbone dihedral angles from cross-correlated spin relaxation

The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei ¹³C, ¹H,¹³CO, ¹⁵N and ¹H^N can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state ¹³CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between ¹H(i)-¹³C(i)/¹⁵N(i)-¹H^N(i) and ¹H(i–1)-¹³C(i–1)/¹⁵N(i)-¹H^N(i) dipole-dipole mechanisms in ¹⁵N,¹³C-labeled proteins. The approach is illustrated with an application to ¹³C,¹⁵N-labeled ubiquitin.     

Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: Distinctive global and residue level features

We present circular dichroism (CD), steady state fluorescence and multidimensional NMR investigations on the equilibrium unfolding of monomeric dynein light chain protein (DLC8) by urea and guanidine hydrochloride (GdnHCl). Quantitative analysis of the CD and fluorescence denaturation curves reveals that urea unfolding is a two-state process, whereas guanidine unfolding is more complex. NMR investigations in the native state and in the near native states created by low denaturant concentrations enabled residue level characterization of the early structural and dynamic perturbations by the two denaturants. Firstly, (15)N transverse relaxation rates in the native state indicate that the regions around N10, Q27, the loop between beta2 and beta4 strands, and K87 at the C-terminal are potential unfolding initiation sites in the protein. Amide and (15)N chemical shift perturbations indicate different accessibilities of the residues along the chain and help identify locations of the early perturbations by the two denaturants. Guanidine and urea are seen to interact at several sites some of which are different in the two cases. Notable among the common interaction site is that around K87 which is in close proximity to W54 on the protein structure, but the interaction modes of the two denaturants are different. The secondary chemical shifts indicate that the structural perturbation by 1M urea is small, compared to that by guanidine which is more encompassing over the length of the chain. The probable (phi, psi) changes at the individual residues have been calculated using the TALOS algorithm. It appears that the helices in the protein are significantly perturbed by guanidine. Further, comparison of the spectral density functions of the native and the two near native states in the two denaturants implicate greater loosening of the structure by guanidine as compared to that by urea, even though the structures are still in the native state ensemble. These differences in the early perturbations of the native state structure and dynamics by the two denaturants might direct the protein along different pathways, as the unfolding progresses on further increasing the denaturant concentration.     

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone¹ H^N, ¹⁵N,¹³ C and¹³ C and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.     

The foldon substructure of staphylococcal nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔG_HX) and the kinetic unfolding and refolding rates (k_op and k_cl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 × 10^− 6 s^− 1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.