The perspectives of studying multi-domain protein folding

Abstract

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two-state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins that often unfold irreversibly under in vitro conditions. This makes folding studies difficult or even impossible. In spite of these problems for many multi-domain proteins, folding has been investigated by classical refolding. Co-translational folding of nascent polypeptide chains when synthesized by ribosomes has also been studied. Single molecule techniques represent a promising approach for future studies on the folding of multi-domain proteins, and tremendous advances have been made in these techniques in recent years. In particular, fluorescence-based methods can contribute significantly to an understanding of the fundamental principles of multi-domain protein folding.

Native and Unfolded States of Phosphoglycerate Kinase Studied by Single‐Molecule FRET

“…[19] With respect to the native state we observe for fully unfolded states of PGK (with GndHCl concentrations above 2 m) a strong structural expansion of the protein which is related to an increase of the mean inter-dye distance R by a factor of two ( Figure 4). Similar results were obtained for PGK by the use of 706 www.chemphyschem.org dynamic light scattering [31] and of FCS, [32] where hydrodynamic radii also increase by a factor of two at high GndHCl concentrations ( Figure S2, Supporting Information). However, in principle, it is of greater interest to elucidate details of the unfolded state under native-like conditions, that is, at low denaturant concentrations.…”

“…Protein folding as well as the stability of this twodomain protein has been studied extensively, in most cases by employing intrinsic tryptophan fluorescence, CD spectroscopy, and dynamic light scattering. [26][27][28][29][30][31][32] In contrast to many other multi-domain proteins, the unfolding transition of PGK induced by guanidine hydrochloride (GndHCl) at elevated temperatures and at low temperatures is highly reversible. [26,31] Due to this fact, PGK has become one of the model systems to study properties of multi-domain protein folding.…”

Protein folding at single-molecule resolution

“…This is important to permit improved means to uncover thermodynamic and time-dependent information from complex single-molecule data. While most single-molecule folding studies have been on relatively simple systems, the majority of the cellular proteome is composed of large, multi-domain proteins that have complex folding behavior and sometimes a significant degree of irreversibility [141]. Single-molecule measurements will continue to target these types of systems, affording substantial advantages in measuring details of the complexity and also minimizing the side-reaction of aggregation.…”

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions, Part 2: Nucleation and Growth Kinetics with Competing Growth Mechanisms

“…3,7 Aggregation pathways have a number of common features across a range of proteins. 2,5,[8][9][10][11][12][13][14][15][16][17][18] Aggregate formation often involves some or all of the following steps, depending on the protein and conditions of interest: monomer unfolding and/ or misfolding, 9,11,17 reversible self-association, 15,16,19 nucleation or creation of the smallest stable (i.e., net irreversible) aggregates, 8,10,13 subsequent growth of soluble aggregates by monomer addition and/ or aggregate-aggregate coalescence or condensation polymerization, [12][13][14]18 and aggregate precipitation and/or macroscopic particle formation. 12 A number of theoretical and experimental models capture some of these steps in the multistep process of aggregation.…”