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Titolo:
CHARACTERIZING THE USE OF PERDEUTERATION IN NMR-STUDIES OF LARGE PROTEINS C-13, N-15 AND H-1 ASSIGNMENTS OF HUMAN CARBONIC-ANHYDRASE-II
Autore:
VENTERS RA; FARMER BT; FIERKE CA; SPICER LD;
Indirizzi:
DUKE UNIV,NMR CTR,BOX 3711 DURHAM NC 27710 DUKE UNIV,MED CTR,DEPT BIOCHEM DURHAM NC 27710 DUKE UNIV,MED CTR,DEPT RADIOL DURHAM NC 27710 BRISTOL MYERS SQUIBB PHARMACEUT RES INST,MACROMOL NMR PRINCETON NJ 08543
Titolo Testata:
Journal of Molecular Biology
fascicolo: 5, volume: 264, anno: 1996,
pagine: 1101 - 1116
SICI:
0022-2836(1996)264:5<1101:CTUOPI>2.0.ZU;2-F
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR-MAGNETIC-RESONANCE; ISOTOPICALLY-ENRICHED PROTEINS; HYDROGEN-BOND NETWORK; SECONDARY STRUCTURE; BACKBONE ASSIGNMENTS; RELAXATION RATES; CHEMICAL-SHIFTS; SEQUENTIAL ASSIGNMENT; IMPROVED SENSITIVITY; CROSS-CORRELATION;
Keywords:
PERDEUTERATION; CARBONIC ANHYDRASE; NMR ASSIGNMENTS; SECONDARY STRUCTURE; ISOTOPE SHIFTS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
73
Recensione:
Indirizzi per estratti:
Citazione:
R.A. Venters et al., "CHARACTERIZING THE USE OF PERDEUTERATION IN NMR-STUDIES OF LARGE PROTEINS C-13, N-15 AND H-1 ASSIGNMENTS OF HUMAN CARBONIC-ANHYDRASE-II", Journal of Molecular Biology, 264(5), 1996, pp. 1101-1116

Abstract

Perdeuteration of all non-exchangeable proton sites can significantlyincrease the size of proteins and protein complexes for which NMR resonance assignments and structural studies are possible. Backbone H-1, N-15, (CO)-C-13, C-13(alpha) and C-13(beta) chemical shifts and aliphatic side-chain C-13 and H-1(N)/N-15 chemical shifts for human carbonicanhydrase II (HCA II), a 259 residue 29 kDa metalloenzyme, have been determined using a strategy based on 2D, 3D and 4D heteronuclear NMR experiments, and on perdeuterated C-13/N-15-labeled protein. To date, HCA II is one of the largest monomeric proteins studied in detail by high-resolution NMR. Of the backbone resonances, 85% have been assigned using fully protonated N-15 and C-3/N-15-labeled protein in conjunction with established procedures based on now standard 2D and 3D NMR experiments. HCA II has been perdeuterated both to complete the backbone resonance assignment and to assign the aliphatic side-chain C-13 and H-1(N)/N-15 resonances. The incorporation of H-2 into HCA II dramatically decreases the rate of C-13 and (HNT2)-H-1 relaxation. This, in turn,increases the sensitivity of several key H-1/C-13/N-15 triple-resonance correlation experiments. Many otherwise marginal heteronuclear 3D and 4D correlation experiments, which are important to the assignment strategy detailed herein, can now be executed successfully on HCA II. Further analysis suggests that, from the perspective of sensitivity, perdeuteration should allow other proteins with rotational correlation times significantly longer than HCA II (tau(c) = 11.4 ns) to be studiedsuccessfully with these experiments. Two different protocols have been used to characterize the secondary structure of HCA II from backbonechemical-shift data. Secondary structural elements determined in thismanner compare favorably with those elements determined from a consensus analysis of the HCA II crystal structure. Finally, having outlineda general strategy for assigning backbone and side-chain resonances in a perdeuterated large protein, we propose a strategy whereby this information can be used to glean more detailed structural information from the partially or fully protonated protein equivalent. (C) 1996 Academic Press Limited

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Documento generato il 04/07/20 alle ore 23:39:52