Time-resolved biophysical methods in the study of protein folding

doi:10.1016/S0959-440X(96)80029-7

Current Opinion in Structural Biology

Volume 6, Issue 5, October 1996, Pages 630-636

https://doi.org/10.1016/S0959-440X(96)80029-7 Get rights and content

Abstract

Many of the biophysical techniques developed to characterize native proteins at equilibrium have now been adapted to the structural and thermodynamic characterization of transient intermediate populations during protein folding. Recent advances in these techniques, the use of novel methods of initiating refolding, and a convergence of theoretical and experimental approaches are leading to a detailed understanding of many aspects of the folding process.

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This review aims to highlight the fundamental mechanism of protein misfolding leading to protein aggregation and associated diseases. It also aims to anticipate novel therapeutic strategies with which to prevent or treat these highly debilitating conditions linked to these pathologies. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. The core cause of over 20 different human diseases which have now been designated as ‘conformational diseases’ including neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson’s disease (PD) etc. A comprehensive study on protein misfolding, aggregation, and the outcomes of the effects of cytotoxic aggregates will lead to understand the aggregation-mediated cell toxicity and serves as a foundation for future research in development of promising therapies and drugs. This review has also shed light on the mechanism of protein misfolding which leads to its aggregation and hence the neurodegeneration. From these considerations, one could also envisage the possibility that protein aggregation may be exploited by nature to perform specific physiological functions in differing biological contexts.
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It is of great interest to determine whether various protein folding routes predominate in the presence of stabilizing osmolytes, because this will imply that the folding routes taken in the cell also rely on osmolytes present within it. Lots of work has been done to characterize denatured states and thermodynamically stable folding intermediates of proteins under various solvent conditions such as organic solvents, high salt concentrations, very high or low pH, low concentrations of strong (guanidinium chloride (GdmCl) and urea) and salt-denaturants [24–39]. The process of folding in case of proteins having more than one domain is a quite complex as different domains and sub-domains fold independently and interact with each other to produce the native conformation [40–44].
Recently, we had published that urea-induced denaturation curves of optical properties of sheep serum albumin (SSA) are biphasic with a stable intermediate that has characteristics of molten globule (MG) state. In this study, we have extended the work by carrying out urea- and guanidinium chloride (GdmCl)-induced denaturations of SSA in the presence of naturally occurring mammalian kidney osmolytes, namely, sorbitol, myo-inositol and glycine betaine. We have observed that all these osmolytes (i) transform this biphasic transition into a co-operative, two-state transition and (ii) increase the stability of the protein in terms of midpoint of denaturation (C_m) and Gibbs free energy change in the absence of both denaturants (ΔG_D⁰). The relative effectiveness of different osmolytes on the stability of SSA follows the order: glycine betaine > myo-inositol > sorbitol. In this paper, we also report that kidney osmolytes destabilize MG state by shifting the equilibrium, native state ↔ MG state toward the left. This study will be helpful in understanding the existence of osmolytes in kidney and their role in folding of kidney proteins soaked with urea.
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The presence of MGs can be revealed through use of extrinsic dyes, such as thioflavin T (ThT), as these dyes become highly fluorescent upon binding to exposed hydrophobic residues (25). Other methods for MG detection include circular dichroism and intrinsic tryptophan fluorescence (26–28). However, these methodologies are unsuitable for studying co-translational MG formation, as ribosome-nascent chain complexes (RNCs) not only contain the emerging polypeptide but also more than 50 ribosomal proteins.
Folding of proteins usually involves intermediates, of which an important type is the molten globule (MG). MGs are ensembles of interconverting conformers that contain (non-)native secondary structure and lack the tightly packed tertiary structure of natively folded globular proteins. Whereas MGs of various purified proteins have been probed to date, no data are available on their presence and/or effect during protein synthesis. To study whether MGs arise during translation, we use ribosome-nascent chain (RNC) complexes of the electron transfer protein flavodoxin. Full-length isolated flavodoxin, which contains a non-covalently bound flavin mononucleotide (FMN) as cofactor, acquires its native α/β parallel topology via a folding mechanism that contains an off-pathway intermediate with molten globular characteristics. Extensive population of this MG state occurs at physiological ionic strength for apoflavodoxin variant F44Y, in which a phenylalanine at position 44 is changed to a tyrosine. Here, we show for the first time that ascertaining the binding rate of FMN as a function of ionic strength can be used as a tool to determine the presence of the off-pathway MG on the ribosome. Application of this methodology to F44Y apoflavodoxin RNCs shows that at physiological ionic strength the ribosome influences formation of the off-pathway MG and forces the nascent chain toward the native state.
2,2,2-Trifluroethanol induces simultaneous increase in α-helicity and aggregation in alkaline unfolded state of bovine serum albumin
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Little work has been done to understand the folding of proteins at alkaline conditions. BSA acquires a partially reversible unfolded state at pH 13.0, devoid of any native structure. Introduction of methanol, ethanol and 2-propanol with the alkaline unfolded protein resulted in β-sheet-like structure formation, and 2,2,2-trifluroethanol found to enhance α-helical conformations with simultaneous increase in aggregation. The extent of secondary and tertiary structure formation is in the order of methanol < ethanol < 2-propanol < 2,2,2-trifluroethanol. Exposure of hydrophobic core of protein molecules in apolar environment of 2,2,2-trifluroethanol seems to promote intermolecular cluster formation. This is one of the very few reports that α-helical structures can also aggregate.
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Time-resolved biophysical methods in the study of protein folding

Abstract

Curr Opin Struct Biol

J Biol Chem

Proc Natl Acad Sci USA

Biochemistry

J Am Chem Soc

Biochemistry

Proc Natl Acad Sci USA

Biochemistry

Proc Natl Acad Sci USA

Are there pathways for protein folding?

J Chem Phys

Finding intermediates in protein folding

Bioessays

High resolution mixer for the study of rapid reactions in solution

Rev Sci Instr

Probing the structure of folding intermediates

Curr Opin Struct Biol

Protein folding triggered by electron transfer

Science

Submillisecond events in protein folding

Proc Natl Acad Sci USA

Direct observation of fast protein folding: the initial collapse of apomyoglobin

Proc Natl Acad Sci USA

Collapse and cooperativity in protein folding

Curr Opin Struct Biol

Nature of the early folding intermediate of ribonuclease A

Biochemistry

Experimental investigation of side chain interactions in early folding intermediates

Fold Des

Kinetics of interaction of partially folded proteins with a hydrophobic dye: evidence that molten globule character is maximal in early folding intermediates

Protein Sci

Characterization of an intermediate in the folding pathway of phosphoglycerate kinase: chemical reactivity of genetically introduced cysteinyl residues during the folding process

Biochemistry

Modification of a specific tyrosine enables tracing of the end-to-end distance during apomyoglobin folding

FEBS Lett

Use of fluorescence energy transfer to characterize the compactness of the constant fragment of an immunoglobin light chain in the early stage of folding

Biochemistry

Small angle scattering studies of protein folding

Curr Opin Struct Biol