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Abstract
The most complex problem in studying multi-state protein folding is the determination of the sequence of formation of protein intermediate states. A far more complex issue is to determine at what stages of protein folding its various parts (secondary structure elements) develop. The structure and properties of different intermediate states depend in particular on these parts. An experimental approach, named μ-analysis, which allows understanding the order of formation of structural elements upon folding of a multi-state protein was used in this study. In this approach the same elements of the protein secondary structure are “tested” by substitutions of single hydrophobic amino acids and by incorporation of cysteine bridges. Single substitutions of hydrophobic amino acids contribute to yielding information on the late stages of protein folding while incorporation of ss-bridges allows obtaining data on the initial stages of folding. As a result of such an μ-analysis, we have determined the order of formation of beta-hairpins upon folding of the green fluorescent protein.
Highlights
Fundamental studies of denaturation and renaturation of small two-state proteins have allowed us to develop experimental approaches for obtaining information on the effect of individual amino acid residues on the protein energy landscape
Having analyzed literature data on the effect of incorporated ss-bridges on the proteins [10,11,12,13,14,15,16] and having studied the folding of several multi-state proteins [12,17,18,19,20], we concluded that the introduction of an ss-bridge affects mainly early intermediate stages upon protein folding, whereas single substitutions of hydrophobic amino acids affect late intermediate stages
Many researchers believe that the polypeptide chain in this state should be packed more or less ‘‘correctly’’, but at the same time there are no tight contacts of amino acid residues
Summary
Fundamental studies of denaturation and renaturation of small two-state proteins have allowed us to develop experimental approaches for obtaining information on the effect of individual amino acid residues on the protein energy landscape (see, e.g. [1,2]). [1,2]) One of such approaches is the phi-analysis using which we can determine amino acid residues included in the so-called ‘‘folding nucleus’’ (i.e. the structured part of the ‘‘transition state’’). This approach consists in measuring and analyzing the folding and unfolding rates of the wild-type protein and its mutant forms (with single amino acid substitutions) at various concentrations of the denaturant [3,4,5]. Experimental studies of complex proteins with stable intermediate states in their folding are rather complicated. The main difficulty lies in the treatment and interpretation of intricate kinetic data, inasmuch as the more the number of different states in protein, the more difficult it is to differentiate them and to appreciate their formation order and mutual influence
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