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.
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(denoted as Y3AW) of the N-terminal domain 1-47 of hirudin, a highly potent thrombin inhibitor, in which Tyr 3 has been replaced by AW.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 corresponding 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.
De Filippis
Vincenzo
V
Department of Pharmaceutical Sciences, University of Padua, via F. Marzolo 5, I-35131 Padua, Italy. vincenzo.defilippis@unipd.it
De Boni
Silvia
S
De Dea
Elisa
E
Dalzoppo
Daniele
D
Grandi
Claudio
C
Fontana
Angelo
A
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism*; D007865:Leeches; D008958:Models, Molecular; D015335:Molecular Probes / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism
D006629:Hirudins; D015335:Molecular Probes; C006657:7-azatryptophan; D014364:Tryptophan; D013917:Thrombin