Sequence-specific sup 1 H NMR assignments and secondary structure in solution of Escherichia coli trp repressor

Journal Article · Tue Jul 10 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00479a002· OSTI ID:6290534

Arrowsmith, C H; Pachter, R; Altman, R B; Iyer, S F; Jardetzky, O ^[1]

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1. Stanford Univ., CA (USA)

Sequence-specific {sup 1}H NMR assignments are reported for the active L-tryptophan-bound form of Escherichia coli trp repressor. The repressor is a symmetric dimer of 107 residues per monomer; thus at 25 kDa, this is the largest protein for which such detailed sequence-specific assignments have been made. At this molecular mass the broad line widths of the NMR resonances preclude the use of assignment methods based on {sup 1}H-{sup 1}H scalar coupling. The assignment strategy centers on two-dimensional nuclear Overhauser spectroscopy (NOESY) of a series of selectively deuterated repressor analogues. A new methodology was developed for analysis of the spectra on the basis of the effects of selective deuteration on cross-peak intensities in the NOESY spectra. A total of 90% of the backbone amide protons have been assigned, and 70% of the {alpha} and side-chain proton resonances are assigned. The local secondary structure was calculated from sequential and medium-range backbone NOEs with the double-iterated Kalman filter method. Since the repressor is a symmetric dimer, long-range intersubunit NOEs were distinguished from intrasubunit interactions by formation of heterodimers between two appropriate selectively deuterated proteins and comparison of the resulting NOESY spectrum with that of each selectively deuterated homodimer. Thus, from spectra of three heterodimers, long-range NOEs between eight pairs of residues were identified as intersubunit NOEs, and two additional long-range intrasubunits NOEs were assigned.

• OSTI ID: 6290534
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:27; 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
BACTERIA
BARYONS
ELEMENTARY PARTICLES
ESCHERICHIA COLI
FERMIONS
GENE REPRESSORS
HADRONS
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEAR MAGNETIC RESONANCE
NUCLEONS
NUCLEOPROTEINS
ORGANIC COMPOUNDS
OVERHAUSER EFFECT
PROTEINS
PROTONS
RESONANCE
TRANSCRIPTION FACTORS