Determination of 3J(H infi supN ,C infi sup? ) coupling constants in proteins with the C?-FIDS method

Abstract

We introduce the C'-FIDS-(1)H,(15)N-HSQC experiment, a new method for the determination of (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling constants in proteins, yielding information about the torsional angle ϕ. It relies on the (1)H,(15)N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three (1)H,(15)N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t(1) and t(2), a second experiment in which they are decoupled exclusively during t(1) and a third one in which they are coupled in t(1) as well as t(2). The last experiment yields an E.COSY-type pattern from which the (2)J(H (infi) (supN) ,C (infi-1) (sup') ) and (1)J(N(i),C (infi-1) (sup') ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the (2)J(H (infi) (supN) ,C (infi-1) (sup') ) coupling, the (3)J(H (infi) (supN) ,C (infi) (sup') ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for (n)J(H(N),C') couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.

Measurement of the Degree of Coupled Isotopic Enrichment of Different Positions in an Antibiotic Peptide by NMR

“…Such information is doubled the sensitivity of this experiment to 15 N by employing a constant time 13 C evolution period of 1/J, resulting in signals of amplitude 1 or 01 for 13 C not coupled or cou-pled to 15 N, respectively, and also included 13 C chemical-ple availability, our method yields reliable measures of the fraction of 13 C coupled to 15 N, for each of the C sites indepenshift evolution in the constant time delay to obtain signal dispersion in 2D (5,6). dently, and for each of the possible 15 N coupling partners, thus providing information on the biosynthetic pathway of A more fundamental limitation on the measurement of coupled isotope incorporation is imposed by unknown and valclavam in S. antibioticus. small J couplings arising either because low gyromagnetic ratio nuclei are involved or because the two sites of inter-METHODS est are connected by multiple bonds.…”

“…HMQC has been used to distinguish between small mole-NMR spectroscopy can provide information as to what cules with and without 15 N at a given position by allowing sites have been labeled with NMR active isotopes, and to 13 C to evolve under coupling to 15 N for a period of 1/2J, what degree (3). NMR can also reveal the fraction of mole-with the result that the signal intensity is either 1 or 0 decules in which a labeled site is close to another labeled pending on whether the 13 C is coupled to 15 N (4).…”

“…small J couplings arising either because low gyromagnetic ratio nuclei are involved or because the two sites of inter-METHODS est are connected by multiple bonds. In this situation, measurement of J by the E.COSY approach ( 7,8 ) is lim-Sample Preparation and NMR Spectroscopy ited somewhat by the need for very high resolution in the indirect dimension in order to resolve couplings to the [ 13 C, 15 N]-labeled valclavam was produced by fermentation of S. antibioticus Tü 1718 (18) in a modified soy meindirectly detected nucleus [ but see ( 9,10 ) ] . Moreover, E.COSY-type methods are complicated when a substantial dium as described elsewhere (19).…”

“…Moreover, E.COSY-type methods are complicated when a substantial dium as described elsewhere (19). The onset of clavam production was detected by imidazole derivatization and UV fraction of the sites to which coupling is to be measured are not labeled, as the signal from these sites falls between assay (20,21) and marked the first of two equal administrations of filtration-sterilized [2-13 C, 15 N] glycine (1 g total, the coupled multiplet components and obscures their splitting. Methods that decrease the linewidths of the signal 7.2 m M; Isotech), 53 and 62 h after innoculation.…”

Pulse Sequences for Measuring Coupling Constants

“…Alternative methods for measuring J couplings include the so-called P-FIDS or C´-FIDS methods Rexroth et al, 1995a) and the ZQ/DQ methods (Rexroth et al, 1995b;Otting, 1997).…”