Pulsed H/D-exchange studies of folding intermediates

doi:10.1016/0959-440X(93)90206-Z

Current Opinion in Structural Biology

Volume 3, Issue 1, February 1993, Pages 84-91

https://doi.org/10.1016/0959-440X(93)90206-Z Get rights and content

Abstract

Pulsed peptide H/D exchange has been used recently to study intermediates on the folding pathways of nine small proteins, in eight laboratories. Patterns of folding behavior are beginning to emerge and some results are quite unexpected. The data are compared with predictions of a molten-globule model of protein folding.

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Discovering universal laws governing protein folding and stability may profoundly impact different aspects of human life, ranging from industrial applications of proteins to medicinal biotechnology for the prevention and treatment of diseases. Despite a considerable number of investigations, including theoretical, computational, and experimental studies on the sequence, structure, and function of proteins, the protein folding problem is still an unanswered question. Protein folding is a fast process that occurs on a time scale of a few microseconds to a few seconds. Folding kinetics depends on multiple factors, including protein sequence and external conditions, and can be studied using various spectroscopic methods combining different unfolding/refolding techniques. In this chapter, we attempt to discuss experimental aspects of protein folding kinetics. The methodology, data analysis, and interpretation of kinetic data in protein folding studies are discussed.
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After that, this species has been recognized for some other proteins (Chamani, Moosavi-Movahedi, Saboury, Gharanfoli, & Hakimelahi, 2003; Kuwajima, 1989; Ptitsyn, 1992). The amount of secondary structure elements in the equilibrium state of several proteins has been characterized using some biophysical techniques such as 2D-HNMR and amide-proton/deuterium transfer (Baldwin, 1993). Recently, we used the hydrophobic interaction notion to mean the total interactions involved in the movement of nonpolar groups from aqueous to a nonpolar milieu.
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Myoglobin is known to show formation of intermediate states under various environmental conditions, in spite of that, this is the first evidence of formation pre-molten globule (PMG) in myoglobin. Polyethylene glycol (PEG) of various molecular sizes shows assorted effects on different proteins. Out of too short and too long PEGs, only PEGs of optimal size interact with proteins leading to change in protein structure that form intermediate state. We are the first one to report the formation of PMG in a protein in the presence of a crowding agent. The PEG-induced intermediate state was characterized by various techniques like absorption, fluorescence, near- and far-UV circular dichroism spectroscopy, ANS binding, and dynamic light scattering measurements to be PMG. Isothermal titration calorimetry and docking studies were further carried out to delineate the mechanism of formation of PMG in myoglobin in physiological conditions. The intermediate formed due to interaction of PEG with myoglobin has physiological implications which are essential to unravel the mystery to solve the massively complicated problems involved in the proper folding of proteins in vivo. Further, outcomes from this study are expected to gain mechanistic insights on the native structure and functions of proteins under in vivo conditions.
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This research aims at showing that deciphering NMR patterns of peptide anisochronous methylene protons (A and B) coupled to an adjacent NH or CH(X) hydrogen may bring novel structural and dynamical information, using for this purpose four l-Histidine-containing peptides and the NMR patterns of Glycyl or β-Alanyl residues or of the Histidyl side-chain, or else of a grafted N-butyl chain. Investigations were carried out along the following lines: ionization constants of functional end groups and speciation curves of protonated species; specific anisochronies and coupling constants assigned to each state of protonation of each compound; base-catalyzed amide proton exchange rate and peptide pK's from DNMR of anisochronous protons. Vicinal coupling constants J_AX and J_BX were found (i) equal along the peptide backbone, suggesting either free rotation about the N–C bond, or, more probably, a torsion angle Φ=90°, and (ii) unequal in the Histidyl side-chain, suggesting an Imidazole-carboxylate interaction preventing free rotation. Peptide pK's span a wide range of values, from 13.63 to 17.48, depending on the parent residue and the characteristics of functional end groups.
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The interdependence of these effects makes it difficult to get both nuclear and kinetic parameters with sufficient precision, depending on the position of the pH kinetic windows and the magnitude of anisochronies in each case. The mechanism of the acid-catalyzed proton exchange in amides has been proposed to involve either N-protonation,7b or O-protonation,32,7h,j or both N- and O-protonations. The application of these proposals to a diamide such as AGHA is an increasingly complex problem (for a discussion, see Appendix A in the Supplementary data), but whatever the exact interpretation, NMR rate constants kNH proved to be proportional to hydronium ion concentration with an acceptable level of accuracy: kNH=kH [H+].
The objective of the present research is to explore the potential of an additional feature of proton 1D-NMR for the study of the properties of peptides in aqueous solution, namely the magnetic non-equivalence of N-methylene protons in linear acyclic peptides induced by an asymmetric carbon atom located nearby. For this exploratory study, we have chosen an l-Alanyl terminal residue adjacent to a Glycyl residue, itself grafted to an optically inactive terminal Histamine unit, i.e., the Histamine-containing pseudopeptide l-Alanyl-Glycyl-Histamine (AGHA). The pH- and temperature-dependence of magnetic non-equivalences of Glycyl and Histamine N-methylene protons was modeled as a weighted mean over four ionization states BH₃, BH₂, BH, B of AGHA molecules found in the pH range explored: pH −0.78 to 12. In parallel, this research has made it possible to assess the pK's governing the speciation curve, the base- and acid-catalyzed proton exchange rates and the pK's of amide bonds.
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Modifications of the overall electrical charge in turn produce variations of the electronic density around hydrogen nuclei, the so-called shielding (by deprotonation) or deshielding (by protonation) effects on NMR chemical shifts.5 In this respect, protonation shifts were used to determine the ionization state of peptides with the pH, as an alternative titration methodology.6 On the other hand, higher NH acidities in proteins have been put in parallel with reduced conformational mobilities,4 possibly accounting for transition from a globular to a random coil form and denaturation phenomena7 when altering the pH from its biological value.
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Pulsed H/D-exchange studies of folding intermediates

Abstract

Biochemistry

Annu Rev Biochem

J Protein Chem

Proteins

Nature

FEBS Lett

Biochemistry

NMR Evidence for an Early Framework Intermediate on the Folding Pathway of Ribonuclease A

Nature

Structural Characterization of Folding Intermediates in Cytochrome c by H-exchange Labelling and Proton NMR

Nature

Early Folding Intermediate of Ribonuclease A

Structure and Stability of Cytochrome c Folding Intermediates

Early Steps in Cytochrome c Folding Probed by Time-resolved Circular Dichroism and Fluorescence Spectroscopy

Biochemistry

Detection and Characterization of a Folding Intermediate in Barnase by NMR

Nature

The Folding of an Enzyme. IV. Structure of an Intermediate in the Refolding of Barnase Analysed by a Protein Engineering Procedure

J Mol Biol

Early Hydrogen-bonding Events in the Folding Reaction of Ubiquitin

The Folding of Hen Lysozyme Involves Partially Structured Intermediates and Multiple Pathways

Nature

Amide Proton Exchanges and Surface Conformation of BPTI in Solution

J Mol Biol

Protein Folding Studied Using Hydrogen Exchange Labeling and Two-dimensional NMR

Annu Rev Biophy Biomol Struct

Characterizing Intermediates in Protein Folding

Curr Biol

Characterizing Protein Folding Intermediates

Curr Biol

Experimental Studies of Pathways of Protein Folding