Perplexing Protein Puzzles

Detta är en avhandling från Department of Biophysical Chemistry, Lund University

Sammanfattning: Protein structure and stability are inherent in the amino acid sequence and governed by non-covalent interactions. The cooperation between forces is, however, perplexing and not well understood. In order to elucidate and predict protein folding and stability, detailed studies of non-covalent interactions are required. Presented in this thesis are detailed studies on electrostatic interactions in proteins and their contribution to protein stability for PGB1, the protein G B1 domain from Streptococcus sp. Electrostatic interactions were investigated both by means of thermal and chemical denaturation at various pH and salt concentrations using CD spectroscopy and differential scanning calorimetry, and by site-specific pKa-value determination using heteronuclear NMR spectroscopy. Investigations of electrostatic interactions both in the folded and unfolded states of the protein were conducted. The results show strong electrostatic coupling between charges in the native protein that was not efficiently screened by salt. In the unfolded state most charge-charge interactions were reduced. Novel methods in analyzing NMR titration data are introduced and used to reveal electrostatic coupling. Moreover, the relative contribution of pKa-shift arising from direct Coulomb interactions, hydrogen bonding and desolvation was elucidated. Using experimentally determined pKa-values of the folded and unfolded states the pH dependent stability could be accurately calculated and compared to denaturation studies. This thesis moreover contains results from research aiming at stabilizing proteins. The same non-covalent interactions govern the stability of an intact protein chain and the affinity between fragments of the chain, which is made use of in the novel method. Reconstituting fragments of a protein were cloned into a split-GFP system where assembly of fused fragments was visualized by green fluorescence. It was shown that the fluorescence intensity correlated to the affinity between fused fragments. In a subsequent study the fluorescence intensity was used to screen for stabilized variants from a small, focused library of PGB1. Increased green fluorescence revealed enhanced stability of intact chain as observed from denaturation studies. Based on thermodynamic principles and in vivo screening the method shows promising results in screening of larger libraries for optimization of protein stability.

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