Proton-proton Dynamics in Proteins by NMR Spectroscopy.

Protein Dynamics;NMR Relaxation;Proton-proton Dynamics;TROSY;Spin Diffusion;Biological Chemistry;Science;Biophysics

Weaver, Daniel SchmittZuiderweg, Erik R P ;

University of Michigan

关键词: Protein Dynamics;    NMR Relaxation;    Proton-proton Dynamics;    TROSY;    Spin Diffusion;    Biological Chemistry;    Science;    Biophysics;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/75930/dswvr_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Proteins are dynamic objects that undergo motion over a wide range of timescales from femtoseconds to hours.Changes in protein dynamics affect important protein operating parameters such as overall protein stability, ligand affinity, allosteric regulation, and catalytic efficiency. In this work, I describe the measurement of proton-proton dynamics in proteins by means of NMR relaxation experiments.The HSQC-TROSY-based ηzK NMR experiment constitutes the methodological basis of this dissertation.This experiment measures the K rate for each amide proton, which is the sum of all individual zero-quantum proton-proton relaxation rates between the amide proton and its proton neighbors.Each individual relaxation rate can be modulated by dynamics along its interproton vector.The ηzK experiment also measures ηz longitudinal amide N/NH DD/CSA cross-correlated relaxation rates.These rates are incorporated into a protocol for measuring protein rotational diffusion tensors.Proton-proton dynamics in proteins are analyzed by comparing K rates with Krig rates calculated for the case of a rigid protein.The ratio of K rates to Krig rates, Q, provides a measurement of distance-weighted average proton-proton dynamics near amide proton sites.Measurement of Ca2+-saturated calmodulin Q parameters in free and peptide-bound forms indicates proton-proton dynamics throughout both forms.Significant rigidification of the proton network takes place on peptide binding.ηzK data on ubiquitin is used to analyze agreement between experimental K rates and published NMR structures of ubiquitin, which all suggest proton-proton dynamics.Based on these observations, correlation between K rates and Krig rates is introduced as a measurement of the quality of a proposed protein structure.Finally, the ηzK experiment is applied to the nucleotide-binding domain (NBD) of the Hsc70 protein.This 44 kDa protein lies beyond the range of practical NMR relaxation spectroscopy prior to development of ηzK experimental techniques; thus, it serves as an explicit test of the method.Experimental K rates of Hsc70 NBD display good correlation with theoretical K rates calculated from three X-ray structures.In conclusion, the ηzK experiment provides a means of measuring proton-proton dynamics in large proteins.These results will broaden our knowledge of protein dynamics and expand the experimental capabilities of the field.