Conformational responses to changes in the state of ionization of titrable groups in proteins

staphylococcal nuclease electrostatics;acid and local unfolding;conformational reorganization and fluctuation;pH sensitivity;NMR relaxation;hydrogen exchange;Biophysics

Richman, Daniel EricGarcia-Moreno, Bertrand E. ;

Johns Hopkins University

关键词: staphylococcal nuclease electrostatics;    acid and local unfolding;    conformational reorganization and fluctuation;    pH sensitivity;    NMR relaxation;    hydrogen exchange;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37949/RICHMAN-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Electrostatic energy links the structural properties of proteins with some of their important biological functions, including catalysis, energy transduction,and binding and recognition. Accurate calculation of electrostatic energy is essential for predicting and for analyzing function from structure. All proteins have many ionizable residues at the protein-water interface. These groups tend to have ionization equilibria (pKa values) shifted slightly relative to their values in water. In contrast, groups buried in the hydrophobic interior usually have highly anomalous pKa values. These shifts are what structure-based calculations have to reproduce to allow examination of contributions from electrostatics to stability, solubility and interactions of proteins.Electrostatic energies are challenging to calculate accurately because proteins are heterogeneous dielectric materials. Any individual ionizable group can experience very different local environments with different dielectric properties. The studies in this thesis examine the hypothesis that proteins reorganize concomitant with changes in their state of ionization. It appears that the pKa value measured experimentally reflects the average of pKa values experienced in the different electrostatic environments corresponding to different conformational microstates. Current computational models fail to sample conformational reorganization of the backbone correctly.Staphyloccocal nuclease (SNase) was used as a model protein in nuclear magnetic resonance (NMR) spectroscopy studies to characterize the conformational rearrangements of the protein coupled to changes in the ionization state of titrable groups. One set of experiments tests the hypothesis that proton binding to surface Asp and Glu side chains drives local unfolding by stabilizing less-native, more water-solvated conformations in which the side chains have normalized pKa values. Increased backbone flexibility in the ps-ns timescale, hydrogen bond (H-bond) breaking on at least the µs timescale, and segmental unfolding were detected near titrating groups as pH decreased into the acidic range. The study identified local structural features and stabilities that modulate the magnitude of electrostatic effects. The data demonstrate that computational approaches to pKa calculations for surface groups must account for local fluctuations spanning a wide range of timescales.A comparative NMR spectroscopy study with the L25K and L125K variants of SNase, each with a Lys residue buried in the hydrophobic interior of the protein, determined locations, timescales, and amplitudes of backbone conformational reorganization coupled with ionization of the buried Lys residues. The L25K protein exhibited an ensemble of local fluctuations of the β barrel in the hundreds of µs timescale and an ensemble of subglobally unfolded β-barrel states in the hundreds of ms timescale with strong pH dependence. The L125K protein exhibited fluctuations of the helix around site 125 in the µs timescale, with negligible pH dependence. These data illustrate the diverse timescales and local structural properties of conformational reorganization coupled to ionization of buried groups, and the challenge to structure-based electrostatics calculations, which must capture these long-timescale processes.