Author/Editor		Kenig, Manca
Title		Študij stabilnosti in hitrosti himernih in točkovnih mutant človeških stefinov
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2002
Volume		str. 89
Language		slo
Abstract		One of the great challenges of science is the prediction of the three-dimensional structure of a protein from its amino acid sequence: the "folding problem". One of the possible insights into the sequence dependence of folding has come from studies of naturally occurring sets of homologous proteins, proteins with similar structure but different thermodynamic and kinetic properties. Human stefins, A and B are very good models for such studies. To delineate the importance of different secondary structure elements on the stability and rate of folding, four chimeric mutants were constructed. The first one, A37B, has 37 amino acid residues of stefin A joined to 61 amino acid residues of stefin B. The second one, B37A, is the opposite: 37 amino acid residues of stefin B joined to 61 amino acid residues of stefin A. Mutant ABA is stefin A, where the a-helix is replaced with that of stefin B and BAB has the a-helix of stefin A in the body of stefin B. All proteins were expressed in E. coli and purified by affmity chromatography and gel filtration. CD and 1 D NMR spectra as well as inhibitory activity were used to confirm the native fold. From equilibrium denaturation experiments and chevron plots, the stability of the proteins was calculated. Stefin A is the most stable, mutants with a-helix of stefin A (A37B and BAB) have higher stability than stefin B. Mutants with a-helix of stefin B (B37A and ABA) are even less stable than stefm B. (Abstract truncated at 2000 characters).
Descriptors		CYSTEINE PROTEINASE INHIBITORS PROTEIN FOLDING CHIMERIC PROTEINS PROTEIN STRUCTURE, SECONDARY ENZYME STABILITY PROTEIN DEFICIENCY AMINO ACID SEQUENCE NUCLEAR MAGNETIC RESONANCE CHROMATOGRAPHY, AFFINITY CHROMATOGRAPHY, GEL BACTERIAL PROTEINS ESCHERICHIA COLI