Overcoming the overlap problem in the assignment of proton NMR spectra of larger proteins by use of three-dimensional heteronuclear proton-nitrogen-15 Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1.beta.

Abstract

The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.

Three‐dimensional solution structure of the HIV‐1 protease complexed with DMP323, a novel cyclic urea‐type inhibitor, determined by nuclear magnetic resonance spectroscopy

“…Stereospecific assignments of Gly a-protons were derived from 3 J~~~w coupling constants, intraresidue NOEs between HN and a-protons, and sequential NOEs between Gly a-protons ( i ) and HN protons ( i + 1). The @-protons of 49 residues in each monomer were stereospecifically assigned using J couplings, obtained from HNHB (Archer et a]., 1991) and HOHAHA-HMQC (Marion et al, 1989) spectra, and intraresidue a-@ distances, estimated from NOEIROE spectra, as described by Clore et al (1991). In some cases, local secondary structure, together with short-range NOEs, were used to assist in making the stereospecific assignments.…”

“…The signals of all Val y-methyl groups were stereospecifically assigned from estimates of 3 J~~c T (Vuister et ai., 1993a), 3 J~~~@ (Archer et a]., 1991), and 3 J~~~a spectra (Vuister & Bax, 1993), and NOEs between Val a-@ and a -y protons. The signals of the Leu &-methyl groups were stereospecifically assigned from NOE patterns observed in 3D NOESY (Marion et al, 1989) and ROESY (Clore et al, 1991), and 4D NOESY (Vuister et al, 1993b) …”

1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution

“…Three-dimensional "N-edited NOESY-HSQC experiments (Fesik & Zuiderweg, 1988;Marion et al, 1989aMarion et al, , 1989dMesserle et al, 1989) were recorded on the HzO sample using mixing times of 75 and 150 ms. Water was suppressed by presaturation. High-power 180" I3C pulses were included in the "N NOESY-HSQC pulse sequence for 13C decoupling during 'H and I5N evolution.…”

Redox‐dependent dynamics of putidaredoxin characterized by amide proton exchange

“…Correlations of nitrogen resonances with C,H and side-chain ' H resonances were made using standard 3D experiments, including 'H,'H-TOCSY/'H,''N-HSQC (TOCSY-HSQC) and 'H,'H-NOESY/'H,''N-HMQC (NOESY-HMQC) (Marion et al, 1989a(Marion et al, , 1989bDriscoll et al, 1990;Fesik & Zuiderweg, 1990). In all 3D experiments, coherences were allowed to evolve on ' H during the first evolution period ( t l ) , followed by appropriate magnetization transfer between protons.…”