טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentBaranovsky Elina
SubjectStructural Studies by Solid State and Solution NMR: Protein-
Protein and Ligand-Protein Interactions
DepartmentDepartment of Chemistry
Supervisor Professor Asher Schmidt
Full Thesis textFull thesis text - English Version


Abstract

Solid state and solution NMR techniques developed within the last decades constitute a powerful means for elucidating structure-function relationships of macromolecules at the molecular level. In this research solution STD NMR spectroscopy (Saturation Transfer Difference) was employed to map the relative strengths of ligand hydrogen atom interactions to wild type and mutant enzymes differing by a single amino acid. Solid state REDOR (Rotational-Echo DOuble-Resonance) NMR spectroscopy was utilized to identify intermolecular interactions between protein units.

The first part of this work addresses the mapping of ligand epitopes upon binding to the xylanase XT6 and to its non-catalytic mutant E159Q. Recently reported ITC measurements employed exclusively this mutant with oligomeric xylan ligands of 2-6 xylan units, under the assumption that the point mutation does not affect ligand binding. We found that replacing the catalytic glutamic acid residue induces substantial alteration of xylobiose binding, hence alluding to its role in binding.

This study exposes the obstacles limiting the scope of applicability of STD methodology (non-uniform relaxation), yet describes a path that is free of these inherent complications. This path is achievable when one ligand is used to "screen" multiple enzymes, as for the wild type vs. a mutant. In this case, the technique probes structural/functional differences solely within the enzyme, and so isolates the specific role of a single amino acid replacement. This mode of STD NMR application constitutes a powerful tool that bares general implications towards the detailed examination of point mutations in the context of structure-function studies.

The second part of this work addresses Zymomonas mobilis Levansucrase that exists in two different structural forms with different catalytic activities. At pH > 7.0 the enzyme is a tightly associated dimer that hydrolyzes sucrose and synthesizes short fructosaccharides. At pH < 6.0, its dimers form ordered microfibrils of ultra-high molecular weight whose catalytic activity switches almost exclusively to the synthesis of levan. The specific interactions that underlie fibril formation, fibril structure and product specificity are still unknown.

Solid state 13C{15N}REDOR NMR spectroscopy was applied to identify the intermolecular interactions involved in levansucrase self-assembly and fibril formation. REDOR was tailored to probe proximities (<5Å) between backbone carbonyls to any 15N label on adjacent dimer. The experimental observations suggested that the fibrilar structure was not preserved under lyophilization and preliminary screening for lyophilization conditions was inconclusive. Future experiments will address this aspect as well as the adequate use of sparse labeling techniques.