|Ph.D Student||Cabri Oshrat|
|Subject||Solid State and Solution NMR Investigation of Structure-|
Function Relationship in Biomacromolecules
|Department||Department of Chemistry||Supervisor||Professor Asher Schmidt|
|Full Thesis text|
In this work, solid state dipolar recoupling NMR methodology (REDOR) complimented by 31P solution NMR and STD NMR experiments are employed in order to investigate the structure-function relationship in biomacromolecules.
KDO8PS from E. Coli (EM) and from hyperthemophile bacteria A. Pyrophilus (EHT) catalyze A5P and PEP condensation to form the essential KDO. EHT presents optimal activity at 85°C and requires metal ion presence. Solid-state NMR characterization of PEP and A5P binding residues EHT indicated: PEP is bound by residues at unique and distinct chemical environment; A5P is bound by residues with generic, heterogeneous chemical environment. Moreover, A5P binding to EHT occurs also at PEP's unique subsite. Hence, two EHT-A5P populations are evidenced: 'proper' and 'reversed' binding.
Two new inhibitors, 2 and 3, of KDO8PS were designed based on earlier NMR study, synthesized and kinetically characterized (Prof. Baasov). Solids and solution NMR show that inhibitors 2 and 3, employ A5P-based inhibition mode with EM, as was observed for inhibitor 1. NMR data suggest that their binding to EHT occurs via mixed 'proper' and 'reversed' binding populations, as exhibited by A5P. In addition, solids REDOR combined with 31P solution NMR, show that binding of 3 to both EM and EHT is tightest compared to 1 and 2. Moreover, its binding is stronger than that of PEP, an observation made for the first time for an intermediate analog.
Specific labeling of native, individual amino acids within a large protein is of high importance. Developing cell-free translation protocol for this purpose in collaboration with Prof. Shoham was initiated. Confirming the occurrence of the tRNA loading step and quantifying its yield posed a major obstacle. Using both solution and solids REDOR NMR we focused on monitoring the first step of the aminoacylation reaction. The NMR data enabled to determine that in-vitro order of reactants addition is crucial: ATP first, lysine second. Correct order yield for single turnover was 47%.
STD designed to determine substrate binding epitope is an additional solution NMR technique to study structure-function of bio-macromolecules. STD NMR was optimized to study xylanase substrates. Xylanase binds xylane and hydrolyzes its b-1-4 xylose backbone. STD was applied to a non-catalytic mutant XT6 with a series of three ligands of increasing length (xylose, xylobiose, xylotriose). Binding epitope of xylobiose was mapped and refined previous crystallographic data; xylose and xylotriose did not give rise to STD effect due to weak and nonspecific binding, respectively.