Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
ASSIGNMENT OF ALIPHATIC SIDE-CHAIN H-1(N) N-15 RESONANCES IN PERDEUTERATED PROTEINS/
Autore:
FARMER BT; VENTERS RA;
Indirizzi:
BRISTOL MYERS SQUIBB PHARMACEUT RES INST,POB 4000 PRINCETON NJ 08543 DUKE UNIV,MED CTR,NMR CTR DURHAM NC 27710
Titolo Testata:
Journal of biomolecular NMR
fascicolo: 1, volume: 7, anno: 1996,
pagine: 59 - 71
SICI:
0925-2738(1996)7:1<59:AOASHN>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
ISOTOPICALLY-ENRICHED PROTEINS; C-13 MAGNETIZATION; NMR-SPECTROSCOPY; BACKBONE AMIDE; SPECTRA; SEQUENCE; DEUTERIATION; SENSITIVITY; STRATEGY; PULSES;
Keywords:
PERDEUTERATION; SIDE-CHAIN H-1(N)/N-15 ASSIGNMENT; HUMAN CARBONIC ANHYDRASE II; 3D NMR;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
45
Recensione:
Indirizzi per estratti:
Citazione:
B.T. Farmer e R.A. Venters, "ASSIGNMENT OF ALIPHATIC SIDE-CHAIN H-1(N) N-15 RESONANCES IN PERDEUTERATED PROTEINS/", Journal of biomolecular NMR, 7(1), 1996, pp. 59-71

Abstract

The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain C-13 assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D N-15/N-15-separated NOESY, as many main-chain and side-chain H-1(N)/N-15 resonances as possible must be assigned. Traditionally, only backbone amide H-1(N)/N-15 resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic sidechain H-1(N)/N-15 resonances to side-chain C-13 resonances with high sensitivity: NH2-filtered 2D H-1-N-15 HSQC (H2N-HSQC), 3D H2N(CO)C-gamma/beta and 3D H2N(COCgamma/beta)C-beta/alpha for glutamine and asparagine side-chain amide groups; 2D refocused H(N-epsilon/zeta)C-delta/epsilon and H(Nepsilon/zetaCdelta/epsilon)C-gamma/delta for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N-epsilon)C-zeta and nonrefocused H(N-epsilon eta)C-zeta for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated C-13-/N-15-labeled human carbonic anhydrase II (H-2-HCA II). Because more than 95% of all side-chain C-13 resonances in H-2-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain H-1(N)/N-15 resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H, protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only H-1(N)-H-1(N), NOE restraints. In these studies, the inclusion of NOE restraints to side-chain HN protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592-9593].

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 12/07/20 alle ore 02:31:00