Multi-State Proteins: Approach Allowing Experimental Determination of the Formation Order of Structure Elements in the Green Fluorescent Protein

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.

Exploring the folding pathway of green fluorescent protein through disulfide engineering

“…Most unfolding time courses were found to fit two exponential phases. Two phases of unfolding were previously observed for Cycle3 GFP . The highest denaturing condition, 60°C and 6 M GuCl, produced single phase data.…”

“…PONDR‐FIT uses primary sequence data and machine learning algorithms to predict intrinsically disordered protein regions; therefore, this approach is unrelated to the hypotheses based on conformational entropy and folding pathway . Additional disulfide bonds and hydrophobic point mutations produce perturbations in calorimetry‐based unfolding kinetics, suggesting a pathway for GFP folding and unfolding …”

“…17 Additional disulfide bonds and hydrophobic point mutations produce perturbations in calorimetry-based unfolding kinetics, suggesting a pathway for GFP folding and unfolding. 21 The folding pathway of GFP has been studied by simulations and experiment. Mutational analysis has mapped regions of the chain that are sensitive to single residue deletions, 22 secondary structure element omission, 23 circular permutation, 24 and noncircular permutation.…”

Predicting protein folding rate change upon point mutation using residue-level coevolutionary information

“…Two‐state proteins fold into their native structures passing through some specific folding intermediates. But determining the folding intermediates and understanding the formation of secondary structural elements are difficult to resolve for multi‐state proteins due to their inherently complex folding process . This complexity of multi‐state folding probably gives rise to several critical interactions, for which earlier we found rCEO better correlates with ln k f in multi‐state proteins, compared to two‐state proteins .…”

Co‐evolutionary constraints of globular proteins correlate with their folding rates

“…Two-state proteins fold into their native 3D structures passing through specific folding intermediates. However, in multi-state proteins, determination of the folding intermediates and development of their secondary structural elements has been proven difficult to resolve due to the inherent complexity of the process [25]. This complexity of multi-state folding might give rise to several critical interactions those no longer persist in native state (therefore, cannot be captured in co analysis), but are reflected in the higher proportion of indirectly contacting and structurally remote CEPs (compared to two-state).…”