Amplification of the Specific Conformational Fluctuation of Proteins by Site-Specific Mutagenesis and Hydrostatic Pressure.

Conformational fluctuation, namely, protein interconversion between different conformations, is crucial to protein function. Outer surface protein A (OspA), comprising N- and C-terminal globular domains linked by a central ß-sheet, is expressed on the surface of Borrelia burgdorferi, the causative agent of Lyme disease, and recognizes the TROSPA receptor in the tick gut. Solution nuclear magnetic resonance studies have shown that the central ß-sheet and C-terminal domain containing TROSPA recognition sites are less stable than the N-terminal domain, revealing an intermediate conformation between the basic folded and completely unfolded proteins. We previously suggested that exposure of receptor-binding sites following denaturation of the C-terminal domain is advantageous for OspA binding to the receptor. Here, we observed amplification of a specific protein fluctuation by pressure perturbation and site-specific mutagenesis. The salt-bridge-destabilized mutant E160D and the cavity-enlarged mutant I243A favored the intermediate. The proportion of the intermediate accounted for almost 100% in E160D at 250 MPa. Strategies using a suitably chosen point mutation with high pressure are generally applicable for amplification of specific conformational fluctuation and potentially improve our understanding of the intermediate conformations of proteins. Knowledge of various conformations, including OspA intermediates, may be useful for designing a vaccine for Lyme disease.

Dynamic aspects of pressure and temperature-stabilized intermediates of outer surface protein A.

Structural characterization of alternatively folded and partially disordered protein conformations remains challenging. Outer surface protein A (OspA) is a pivotal protein in Borrelia infection, which is the etiological agent of Lyme disease. OspA exists in equilibrium with intermediate conformations, in which the central and the C-terminal regions of the protein have lower stabilities than the N-terminal. Here, we characterize pressure- and temperature-stabilized intermediates of OspA by nuclear magnetic resonance spectroscopy combined with paramagnetic relaxation enhancement (PRE). We found that although the C-terminal region of the intermediate was partially disordered, it retains weak specific contact with the N-terminal region, owing to a twist of the central ß-sheet and increased flexibility in the polypeptide chain. The disordered C-terminal region of the pressure-stabilized intermediate was more compact than that of the temperature-stabilized form. Further, molecular dynamics simulation demonstrated that temperature-induced disordering of the ß-sheet was initiated at the C-terminal region and continued through to the central region. An ensemble of simulation snapshots qualitatively described the PRE data from the intermediate and indicated that the intermediate structures of OspA may expose tick receptor-binding sites more readily than does the basic folded conformation.

Paramagnetic relaxation enhancement-assisted structural characterization of a partially disordered conformation of ubiquitin.

Nuclear magnetic resonance (NMR) is a powerful tool to study three-dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N1 and N2 and plus another folded conformation, I, in which some amide group signals of residues 33-41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well-converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure-induced loss of backbone amide group signals at residues 28, 33, 36, and 39-41 like the wild-type, with a similar but smaller effect on CαH and CßH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N2 conformation, although the amide groups at residues 33-41 have more heterogeneous conformations and more contact with water, which differ from the N1 and N2 states. The PRE-assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.

How internal cavities destabilize a protein.

Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified "invisible" folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å-3.

Rational design using sequence information only produces a peptide that binds to the intrinsically disordered region of p53.

Intrinsically disordered regions (IDRs) of proteins are involved in many diseases. The rational drug design against disease-mediating proteins is often based on the 3D structure; however, the flexible structure of IDRs hinders the use of such structure-based design methods. Here, we developed a rational design method to obtain a peptide that can bind an IDR using only sequence information based on the statistical contact energy of amino acid pairs. We applied the method to the disordered C-terminal domain of the tumor suppressor p53. Titration experiments revealed that one of the designed peptides, DP6, has a druggable affinity of ~1 µM to the p53 C-terminal domain. NMR spectroscopy and molecular dynamics simulation revealed that DP6 selectively binds to the vicinity of the target sequence in the C-terminal domain of p53. DP6 inhibits the nonspecific DNA binding of a tetrameric form of the p53 C-terminal domain, but does not significantly affect the specific DNA binding of a tetrameric form of the p53 core domain. Single-molecule measurements revealed that DP6 retards the 1D sliding of p53 along DNA, implying modulation of the target searching of p53. Statistical potential-based design may be useful in designing peptides that target IDRs for therapeutic purposes.

Water-Protein Interactions Coupled with Protein Conformational Transition.

Conformational fluctuations of proteins are crucially important for their functions. However, changes in the location and dynamics of hydrated water in many proteins accompanied by the conformational transition have not been fully understood. Here, we used phase-modulated clean chemical exchange NMR approach to investigate pressure-induced changes in water-to-amide proton exchange occurring at sub-second time scale. With the transition of ubiquitin from its native conformation (N1) to an alternative conformation (N2) at 250 MPa, proton exchange rates of residues 32-35, 40-41, and 71, which are located at the C-terminal side of the protein, were significantly increased. These observations can be explained by the destabilization of the hydrogen bonds in the backbone and partial exposure of those amide groups to solvent in N2. We conclude that phase-modulated clean chemical exchange NMR approach coupled with pressure perturbation will be a useful tool for investigations of more open and hydrated protein structures.

Analysis of O2-binding Sites in Proteins Using Gas-Pressure NMR Spectroscopy: Outer Surface Protein A.

Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O2-binding sites by O2-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer ß-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O2-induced PREs for amide protons located around the largest cavity and at the central ß-sheet. We suggested three potential O2-accessible sites in the protein based on the 1/r6 distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central ß-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O2 generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O2 within the largest cavity, egress of O2 from the cavity, and ingress of O2 in the surface crevice of the ß-sheet. These molecular dynamics simulation results qualitatively explained the O2-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands.