Protein Science Attend a BioResearch Product Faire
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Protein Science (2004), 13:1489-1502. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by De Filippis, V.
Right arrow Articles by Fontana, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Filippis, V.
Right arrow Articles by Fontana, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1–47 of hirudin as a probe of hirudin folding and thrombin recognition

Vincenzo De Filippis1,2, Silvia De Boni1,3, Elisa De Dea1, Daniele Dalzoppo1, Claudio Grandi1 and Angelo Fontana1,2

1 Department of Pharmaceutical Sciences and
2 CRIBI Interdepartmental Research Center for Innovative Biotechnology, University of Padua, I-35131 Padua, Italy

(RECEIVED December 4, 2003; FINAL REVISION February 17, 2004; ACCEPTED February 18, 2004)



Abstract

7-Azatryptophan (AW), a noncoded isostere of tryptophan (W), possesses interesting spectral properties. In particular, the presence of a nitrogen atom at position 7 in the indolyl nucleus of AW results in a red shift of the absorption maximum and fluorescence emission by 10 and 46 nm, respectively, compared to W. In the present work, we report the chemical synthesis and the conformational and functional characterization of an analog (denoted as Y3AW) of the N-terminal domain 1–47 of hirudin, a highly potent thrombin inhibitor, in which Tyr 3 has been replaced by AW. The results obtained were compared with those of the cooresponding Y3W analog. We found that the replacement W -> AW reduces affinity for thrombin by 10-fold, likely because of the lower hydrophobicity of AW compared with that of W. Measurements of the resonance energy transfer effect, which was observed between Tyr13 and the amino acid at position 3 upon disulfide-coupled folding, demonstrate that AW behaves as a better energy acceptor than W for studying protein renaturation. The interaction of Y3AW with thrombin was studied by exciting the sample at 320 nm and recording the change in fluorescence of Y3AW on binding to the enzyme. Our results indicate that the fluorescence of AW of hirudin 1–47 in the Y3AW–thrombin complex is strongly quenched, possibly because of the presence of two structural water molecules at the hirudin–thrombin interface that can promote the nonradiative decay of AW in the excited state. The data herein reported demonstrate that the incorporation of AW can be of broad applicability in the study of protein folding and protein–protein interaction.

Keywords: hirudin; thrombin; 7-azatryptophan; noncoded amino acids; anticoagulants; folding; spectroscopy

Abbreviations: 7AI, 7-azaindole • a.m.u., atomic mass units • AW, (L)-7-azatryptophan • W, (L)-tryptophan • BSA, bovine serum albumin • CD, circular dichroism • Cha, cyclohexylalanine • ChCl, choline chloride • DTT, dithiothreitol • EDT, ethanedithiol • ESI, electrospray ionization • FLEC, (+)-1–(9-fluorenyl)ethylchloroformate • Fmoc, 9-fluorenyl-methyl-oxycarbonyl • Fmoc-ONSu, 9-fluorenyl-methyloxycarbonyl-O-N-hydroxy-succinimide • FPLC, fast protein liquid chromatography • FT-IR, Fourier transformed infrared • HBTU, 2–(1H-benzotriazol-1-yl-)-1,1,3,3-tetrameth-yluronium hexafluorophosphate • HOBt, 1-hydroxybenzotriazole • HPLC, high-pressure liquid chromatography • {beta}-ME, {beta}-mercaptoethanol • MES, morpholinoethanesulfonic acid • NMR, nuclear magnetic resonance • PEG, polyethylene glycol • RP, reverse-phase • TEA, triethylamine • TFA, trifluoroacetic acid • UV, ultraviolet • TOF, time of flight • Y3AW, synthetic analog of the hirudin HM2 fragment 1–47 in which Tyr 3 has been replaced by L-7-azatryptophan • Y3W, synthetic analog of the hirudin HM2 fragment 1–47 in which Tyr 3 has been replaced by tryptophan • HM2, hirudin variant isolated from the leech Hirudinaria manillensis

Reprint requests to: Vincenzo De Filippis, Department of Pharmaceutical Sciences, University of Padua, via F. Marzolo 5, I-35131 Padua, Italy; e-mail: vincenzo.defilippis{at}unipd.it; fax: 39-049-827-5366.

3 Present address: Department of Pharmaceutical Chemistry, University of Jena, Philosophenweg 14, 07743 Jena, Germany.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03542104.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Protein Sci.Home page
V. De Filippis, A. Draghi, R. Frasson, C. Grandi, V. Musi, A. Fontana, and A. Pastore
o-Nitrotyrosine and p-iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes
Protein Sci., July 1, 2007; 16(7): 1257 - 1265.
[Abstract] [Full Text] [PDF]

Home page
 Protein Sci.Home page
V. De Filippis, R. Frasson, and A. Fontana
3-Nitrotyrosine as a spectroscopic probe for investigating protein protein interactions.
Protein Sci., May 1, 2006; 15(5): 976 - 986.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2004 by The Protein Society.