Development and application of solution NMR methodology for studying biomolecular structure and dynamics
MetadataShow full item record
This thesis focuses on the development and application of solution state NMR methodology towards studying biomolecular structure and dynamics. In chapter 1, development of slice selective (SS) NMR approach for rapid data acquisition is introduced. In this methodology reduction of total experiment time is achieved by following multiplexing approach such that various portions of the sample are used for data collection in a time-staggered fashion. The concept is exemplified by implementing SS methodology to 2D [ 13 C Me1 H] constant time HMQC and 2D [ 13 C Me / 15 N, 1 H] time shared HMQC NMR experiments that are motivated towards acquiring real time 2D NMR data. In chapter 2, aromatic side-chain dynamics in proteins are probed with residual dipolar couplings (RDCs). Novel insights into aromatic ring flip dynamics are derived by comparing experimentally measured aromatic RDCs with theoretically predicted values based on high resolution crystal structures. Avenues to use aromatic RDCs to estimate upper bounds for lowly populated conformational excited states are explored. The application of aromatic RDCs to assess the sampling accuracy of existing in silico approaches, such as molecular dynamics simulations, is discussed. In chapter 3, solution state protein structure determination of North-east Structural Genomics consortium (NESG) targets EwR120 (15 kDa protein encoded in the genome of Erwinia carotovora ) and OR188 (11 kDa protein, de novo design out-reach target) are discussed. The high-throughput protocol for NMR structure determination implemented and optimized by NESG was adopted. EwR120 structure serves the purpose of structural coverage of protein families for homology modelling and OR188 structure validates and refines the current de novo designing protocols that could facilitate our understanding of protein structure and stability. 2D NMR based thermal stability studies of lipoprotein SR500A is discussed. Finally in chapter 4, support offered to the methodology development of novel NMR experiments motivated towards rapid data acquisition is discussed. In the first portion, the concept of G-matrix Fourier Transform (GFT) is applied to efficiently assign the sugar spin systems present in nucleic acids and is exemplified with 30-nucleotide RNA as an example. The final portion discusses the performance of single-point (SP) mode of acquisition to efficiently measure one-bond (scalar/residual dipolar) couplings for proteins and is demonstrated with two standard protein samples, isotropic ubiquitin and partially aligned Z-domain.