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Titolo:
TRANSIENT HYDROGEN-BONDS IDENTIFIED ON THE SURFACE OF THE NMR SOLUTION STRUCTURE OF HIRUDIN
Autore:
SZYPERSKI T; ANTUCH W; SCHICK M; BETZ A; STONE SR; WUTHRICH K;
Indirizzi:
ETH ZURICH,INST MOLEK BIOL & BIOPHYS CH-8093 ZURICH SWITZERLAND ETH ZURICH,INST MOLEK BIOL & BIOPHYS CH-8093 ZURICH SWITZERLAND UNIV CAMBRIDGE,CTR MRC,DEPT HAEMATOL CAMBRIDGE CB2 2QH ENGLAND
Titolo Testata:
Biochemistry
fascicolo: 31, volume: 33, anno: 1994,
pagine: 9303 - 9310
SICI:
0006-2960(1994)33:31<9303:THIOTS>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR-MAGNETIC-RESONANCE; NAJA-MOSSAMBICA-MOSSAMBICA; RECOMBINANT DESULFATOHIRUDIN; DIRECTED MUTAGENESIS; PROTEIN STRUCTURES; DISTANCE GEOMETRY; TITRATION SHIFTS; AQUEOUS-SOLUTION; INHIBITOR-IIA; THROMBIN;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
37
Recensione:
Indirizzi per estratti:
Citazione:
T. Szyperski et al., "TRANSIENT HYDROGEN-BONDS IDENTIFIED ON THE SURFACE OF THE NMR SOLUTION STRUCTURE OF HIRUDIN", Biochemistry, 33(31), 1994, pp. 9303-9310

Abstract

Recombinant desulfatohirudin retains largely the thrombin-inhibitory activity of natural hirudin from Hirudo medicinalis and causes at mostminimal immune response in humans. With regard to potential pharmaceutical applications it is of interest to further investigate the structural basis of hirudin functions. In this paper transient hydrogen bonds between backbone amide protons and side-chain carboxylates on the protein surface of desulfatohirudin (variant 1) have been identified using two-dimensional H-1 NMR experiments and site-directed mutagenesis. The analysis of pH titration curves measured with NMR enabled the determination of the pK values of all 13 carboxylates, and downfield shifts larger than 0.2 ppm arising from weak bonding interactions with carboxylates were observed for the amide protons of Gly 25, Ser 32, Glu 35, and Cys 39. For these backbone amide protons virtually identical titration parameters were observed in intact desulfatohirudin and the mutant, truncated hirudin(1-51), demonstrating that the hydrogen bond accepters are located in the N-terminal polypeptide segment 1-51. The hydrogen bonds Gly 25 NH-Glu 43 delta COO-, Ser 32 NH-Glu 35 delta COO-, Glu 35 NH-Asp 33 gamma COO-, Glu 35 NH-Glu 35 delta COO-, and Cys 39 NH-Glu 17 delta COO- were identified by considering spatial proximity in the NMR solution structure of hirudin(1-51), and comparing the pK values for the amide protons and the carboxylates in desulfatohirudin and the mutants hirudin(E43Q), hirudin(E35Q), hirudin(D33N) and hirudin(E17A). Comparative structure calculations with and without distance constraints for these hydrogen bonds showed that although they are all compatible with the NMR solution structure, these hydrogen bonds are transient dynamic features of the protein surface which, with the sole exception of Cys 39 NH-Glu 17 delta COO-, would not have been detected in a conventional NMR structure determination. Of special interest is the clear-cut information obtained on the fact that the lifetimes of the dynamic ''bifurcated'' hydrogen-bonding interactions of the amide proton of Glu 35 are in the millisecond time range or shorter.

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Documento generato il 21/10/20 alle ore 14:49:06