Functional dynamics of the anti-HIV lectin OAA and NMR methodology for the study of protein dynamics

by Marta Carneiro
Doctoral thesis
Date of Examination:2015-11-18
Date of issue:2016-01-26
Advisor:Prof. Dr. Christian Griesinger
Referee:Prof. Dr. Jörg Enderlein
Referee:Dr. Tim Grüne
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Markus Zweckstetter
Referee:Prof. Dr. Bert De Groot
Persistent Address: http://dx.doi.org/10.53846/goediss-5486

 

 

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Abstract

English

Specific recognition between biomolecules is at the basis of all biological processes, and a mechanistic description of molecular recognition is crucial for a detailed understanding of these processes. Two limiting models are currently discussed in the context of molecular recognition: (i) induced-fit, which postulates that conformational changes between free and bound states are the result of the binding interaction and (ii) conformational selection, according to which the binding interaction selects one of the multiple conformers that pre-exist in equilibrium in the absence of binding partner. A necessary (but not sufficient) condition to demonstrate a conformational selection scenario is the characterization of conformational substates in the absence of binding partner comparable to the conformations seen for the bound forms. NMR spectroscopy is a powerful technique for studying conformational heterogeneity in solution, given its sensitivity to a broad range of motional timescales in solution with atomic resolution. The first part of this thesis is dedicated to the study of functional dynamics of the lectin OAA, aimed at the elucidation of the molecular recognition mechanism underlying its anti-HIV activity, which stems from binding to high-mannose glycans on the viral envelope glycoproteins. Previously determined X-ray crystallography structures identified a distinct conformational change between the free and sugar-bound protein. By using a variety of NMR methods we show that both sugar-free and sugar-bound conformations are conformational substates of the free protein. Further, our results indicate that the sugarbound conformation is highly populated even in the absence of sugar, suggesting that recognition of high-mannose glycans by OAA proceeds by conformational selection within the ground state. These insights may guide further optimization and/or development of preventive anti-HIV therapeutics. The second part of the thesis is concerned with the development of new strategies aimed at extending the efficacy and accuracy of two NMR methods frequently used to investigate lowly populated conformational states. We show that the demanding experimental time required by exchange-mediated saturation transfer experiments can be reduced by two-fold by making use of Fourier transform and linear prediction. We also demonstrate that the simultaneous analysis of data collected with at least two radiofrequency field strengths is necessary for extracting reliable exchange parameters from these experiments. Additionally, we present a method for the identification of dynamic clusters based on model selection using the Akaike information criterion. The efficiency of the method is discussed in the context of synthetic CPMG relaxation dispersion data, but the principles outlined here can be easily applied on the analysis of a variety of experiments.
Keywords: NMR; Structural Biology; Conformational Selection; Anti-HIV lectin; OAA; Oscillatoria agardhii agglutinin; Molecular Recognition; Protein dynamics
 
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