Highly Fluctuating Protein Structures Revealed by Variable-Pressure Nuclear Magnetic Resonance

Abstract

Although our knowledge of basic folded structures of proteins has dramatically improved, the extent of our corresponding knowledge of higher-energy conformers remains extremely slim. The latter information is crucial for advancing our understanding of mechanisms of protein function, folding, and conformational diseases. Direct spectroscopic detection and analysis of structures of higher-energy conformers are limited, particularly under physiological conditions, either because their equilibrium populations are small or because they exist only transiently in the folding process. A new experimental strategy using pressure perturbation in conjunction with multidimensional NMR spectroscopy is being used to overcome this difficulty. A number of rare conformers are detected under pressure for a variety of proteins such as the Ras-binding domain of RalGDS, beta-lactoglobulin, dihydrofolate reductase, ubiquitin, apomyoglobin, p13(MTCP1), and prion, which disclose a rich world of protein structure between basically folded and globally unfolded states. Specific structures suggest that these conformers are designed for function and are closely identical to kinetic intermediates. Detailed structural determination of higher-energy conformers with variable-pressure NMR will extend our knowledge of protein structure and conformational fluctuation over most of the biologically relevant conformational space.

Computing the stability diagram of the Trp-cage miniprotein

“…The notion that proteins under native conditions are only ''marginally stable'' (7) seems to be an important requirement for their ability to explore different conformational substates (19) and hence for protein function. The application of high hydrostatic pressure (20) has been shown to be able shift the equilibrium of conformational states (21,22), promoting denaturation (23) but also altering the native state (24) and modifying protein-protein interaction (20). Pressure effects on protein structure appear to be determined mostly by changing the balance between hydrophilic and hydrophobic interactions (25)(26)(27).…”

Change in local dynamics of bacteriorhodopsin with retinal isomerization under pressure as studied by fast magic angle spinning NMR

“…9,35 As the hydrogen-bond distance between H and O (H O) in the protein structure changed upon the application of pressure, inducing changes in molar volume, the volume change induced by pressure was attributed to hydration. 36 It is concluded that the hydrated condition of bR was retained, as evidenced by the unchanging line widths (Figures 1-3), while the NMR signals of bR dehydrated by lyophilization were clearly broadened. 37 It is suggested that the pressure caused by the fast MAS experiments induces the isomerization of retinal and modulates the hydrogen-bond network in bR, and consequently, a change in the motion of the residues located in the vicinity of retinal is induced.…”

Ceramic cells for high pressure NMR spectroscopy of proteins

“…However, our autoclave is devised also to reduce the length of the ceramic cell as much as possible. Teflon burst protections are also used earlier in the quartz cell system [1,2,5,16]. This is shown in Fig.…”

“…With high pressure NMR spectroscopy it is possible to obtain novel information about protein biochemistry and biophysics at atomic resolution that is difficult or impossible to obtain with other methods [1][2][3]. By observing the pressure response of proteins one obtains not only data on the local and global mechanical and dynamical properties of the macromolecule [4] but can also shift conformational equilibria and stabilize folding and functional intermediates [5][6][7][8].…”