Hydrogen exchange kinetics in a membrane protein determined by sup 15 N NMR spectroscopy: Use of the INEPT (insensitive nucleus enhancement by polarization transfer) experiment to follow individual amides in detergent-solubilized M13 coat protein

Journal Article · Tue Jul 03 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00478a027· OSTI ID:6229202

Henry, G D; Sykes, B D ^[1]

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1. Univ. of Alberta, Edmonton (Canada)

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous {sup 1}H nuclear magnetic resonance (NMR) study, multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow kinetic sets containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10{sup 5}-fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein the authors use {sup 15}N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiments can be used to transfer magnetization to the {sup 15}N nucleus from a coupled proton; when {sup 15}N-labeled protonated protein is dissolved in {sup 2}H{sub 2}O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H{sup +} and OH{sup {minus}} ions. The time-dependent exchange-out experiment is suitable for slow exchange rates (k{sub ex}). The INEPT experiment was also adapted to measure some of the more rapidly exchanging amides in the coat protein using either saturation transfer from water or exchange effects on the polarization transfer step itself. The results of all of these experiments are consistent with previous models of the coat protein in which a stable segment extends from the hydrophobic membrane-spanning region through to the C-terminus, whereas the N-terminal region is undergoing more extensive dynamic fluctuations.

• OSTI ID: 6229202
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:26; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
AMIDES
AMINO ACIDS
BACTERIA
BACTERIOPHAGES
BARYONS
BIOCHEMICAL REACTION KINETICS
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
ELEMENTARY PARTICLES
ESCHERICHIA COLI
EVEN-ODD NUCLEI
FERMIONS
GLYCINE
GROWTH
HADRONS
HYDROXY ACIDS
ISOTOPES
KINETICS
LEUCINE
LIGHT NUCLEI
MAGNETIC RESONANCE
MEMBRANE PROTEINS
MICROORGANISMS
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEONS
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASITES
PEPTIDES
POLARIZATION
POLYPEPTIDES
PROTEINS
PROTONS
REACTION KINETICS
RESONANCE
STABLE ISOTOPES
TIME DEPENDENCE
TYROSINE
VALINE
VIRUSES