Protein folding kinetics by combined use of rapid mixing techniques and NMR observation of individual amide protons

Journal Article · Mon Sep 01 04:00:00 EDT 1986 · Proteins; (United States)

DOI:https://doi.org/10.1002/prot.340010107· OSTI ID:6994803

Roder, H; Wuethrich, K

A method to be used for experimental studies of protein folding introduced by Schmid and Baldwin, which is based on the competition between amide hydrogen exchange and protein refolding, was extended by using rapid mixing techniques and /sup 1/H NMR to provide site-resolved kinetic information on the early phases of protein structure acquisition. In this method, a protonated solution of the unfolded protein is rapidly mixed with a deuterated buffer solution at conditions assuring protein refolding in the mixture. This simultaneously initiates the exchange of unprotected amide protons with solvent deuterium and the refolding of protein segments which can protect amide groups from further exchange. After variable reaction times the amide proton exchange is quenched while folding to the native form continues to completion. By using /sup 1/H NMR, the extent of exchange at individual amide sites is then measured in the refolded protein. Competition experiments at variable reaction times or variable pH indicate the time at which each amide group is protected in the refolding process. This technique was applied to the basic pancreatic trypsin inhibitor, for which sequence-specific assignments of the amide proton NMR lines had previously been obtained. For eight individual amide protons located in the beta-sheet and the C-terminal alpha-helix of this protein, apparent refolding rates in the range from 15 s-1 to 60 s-1 were observed. These rates are on the time scale of the fast folding phase observed with optical probes.

• Research Organization: Institut fuer Molekularbiologie und Biophysik, Zuerich (Switzerland)
• OSTI ID: 6994803
• Journal Information: Proteins; (United States), Journal Name: Proteins; (United States) Vol. 1:1; ISSN PSFGE
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMIDES
BARYONS
BIOCHEMICAL REACTION KINETICS
DEUTERIUM
ELEMENTARY PARTICLES
FERMIONS
HADRONS
HYDROGEN ISOTOPES
IN VITRO
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MOLECULAR STRUCTURE
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
PROTONS
REACTION KINETICS
RESONANCE
SPECTRA
STABLE ISOTOPES
TRACER TECHNIQUES