DEVELOPMENT AND APPLICATION OF NUCLEAR MAGNETIC RESONANCE TECHNIQUES FOR THE STUDY OF PROTEIN DYNAMICS

Non-ribosombal peptide synthetase;peptidyl carrier protein;linker;covariance;accordion;nuclear magnetic resonance;relaxation;four-dimensional spectra;Biophysics

Harden, Bradley JamesBader, Joel S. ;

Johns Hopkins University

关键词: Non-ribosombal peptide synthetase;    peptidyl carrier protein;    linker;    covariance;    accordion;    nuclear magnetic resonance;    relaxation;    four-dimensional spectra;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/40830/thesis_v3.docx?sequence=2&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The dynamic nature of proteins is a topic of increasing importance in the field of structural biology. Nuclear magnetic resonance (NMR) experiments that characterize protein dynamics offer an unparalleled level of insight into such motional processes. However, a multitude of challenges impede broad application of the technique. The process of assigning each NMR signal to its corresponding atom remains difficult in crowded spectra. Furthermore, lengthy experiments and laborious data analysis can curtail motivations to perform NMR dynamics experiments. In this work, I have developed improvements to two underutilized classes of NMR techniques, and I have applied NMR experiments to study dynamic interactions in an important class of enzymatic systems. I implemented novel covariance NMR processing methods that help minimize artifacts, including a pre-processing spectral derivative that suppresses false positive artifacts stemming from near degeneracy between signals and a post-processing element-wise multiplication step that eliminates coincidental correlations. I applied the improvements to develop a series of novel four-dimensional covariance spectra that streamline the assignment of signals in crowded NMR spectra. I developed a software application to simplify and improve analysis of data acquired with accordion spectroscopy, a technique that can reduce the time required to perform NMR relaxation measurements. I established an approach to fitting accordion NMR data that maximizes accuracy in cases of strong overlap while also ensuring optimal measurement precision. I applied NMR dynamics experiments to study the interaction between a peptidyl carrier protein and its linker regions within a megadalton non-ribosomal peptide synthetase assembly. I found that interaction between the linker region and the carrier protein core stabilized the latter by a factor of six and modulates its fast, ps-ns time-scale motions. This modulation reveals a dynamic allosteric network that may provide communication between a site of post-translational modification and a linker region to help maneuver the carrier protein during synthesis.