Heteronuclear 3D NMR and isotopic labeling of calmodulin. Towards the complete assignment of the 1H NMR spectrum.

نویسندگان

  • M Ikura
  • D Marion
  • L E Kay
  • H Shih
  • M Krinks
  • C B Klee
  • A Bax
چکیده

New methods are described that permit detailed analysis of the NMR spectra of calmodulin, an alpha-helical protein with a molecular weight of 16.7 kD. Two complementary approaches have been used: uniform labeling with 15N and labeling of specific amino acids with either 15N or 13C. It is demonstrated that uniform 15N labeling permits the recording of sensitive three-dimensional (3D) NMR spectra that show far better resolution than their conventional two-dimensional analogs. Selective 15N labeling of amino acids can be used for identifying the type of amino acid, providing information that is essential for the analysis of the 3D spectra. Simultaneous selective labeling with both 15N and 13C can provide a number of unique backbone assignments from which sequential assignment can be continued.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy.

The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (153 residues, Mr = 17,400) has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed ...

متن کامل

A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin.

A novel approach is described for obtaining sequential assignment of the backbone 1H, 13C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (approximately 95%) labeled with 15N and 13C. Sequential assignment of the backbone residues by standard methods was not possible because of the very narrow chemical shift distribution rang...

متن کامل

Dipolar filtered 1H-13C heteronuclear correlation spectroscopy for resonance assignment of proteins.

Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving 13C and 15N spins have been developed for this purpose, H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular 1H-13C heteronuclear correlation (HETCOR) experim...

متن کامل

Through-Bonds and Through-Space Solid-State NMR Correlations at Natural Isotopic Abundance: Signal Assignment and Structural Study of Simvastatin

Through-bond and through-space solid-state NMR correlation experiments at natural isotopic abundance (refocused cross-polarization incredible natural abundance double quantum transfer experiment (CPINADEQUATE), 1H-13C HETCOR, 1H-13C magic-angle spinning J heteronuclear multiple-quantum coherence (MAS-J-HMQC)) are presented on a moderately sized molecule of simavastatin (C25H38O5). Refocused 13C...

متن کامل

Specific Isotopic Labeling of Methyl Groups has Extended the Molecular Weight Limits for NMR Studies of Protein Structure and Dynamics

Nuclear magnetic resonance (NMR) spectroscopy has emerged as the preeminent tool in solution studies of protein structure1-3, dynamics1, 4-10, and intermolecular interactions11-14. A key limitation, however, was that only relatively small proteins could be studied. Recent advances in isotope labeling15-17, the development of TROSY-based methods18, and the advent of cryogenic probes19 have exten...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • Biochemical pharmacology

دوره 40 1  شماره 

صفحات  -

تاریخ انتشار 1990