Tryptophan mutants of human C5a anaphylatoxin: A fluorescence anisotropy decay and energy transfer study

M. Federwisch; A. Wollmer; M. Emde; T. Stühmer; T. Melcher; A. Klos; J. Köhl; W. Bautsch

doi:10.1016/0301-4622(93)80017-D

Tryptophan mutants of human C5a anaphylatoxin: A fluorescence anisotropy decay and energy transfer study

M. Federwisch, A. Wollmer^*, M. Emde, T. Stühmer, T. Melcher, A. Klos, J. Köhl, W. Bautsch

^*Korrespondierende/r Autor/-in für diese Arbeit

Institut für Systemische Entzündungsforschung

11 Zitate (Scopus)

Abstract

Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys²⁷ for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp²]rhC5a > [Trp⁶⁴]rhC5a > [Trp⁷⁰]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp⁷⁰ to the 1,5-AEDANS group at Cys²⁷ yielded a distance distribution of 2.4 ± 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.

Originalsprache	Englisch
Zeitschrift	Biophysical Chemistry
Jahrgang	46
Ausgabenummer	3
Seiten (von - bis)	237-248
Seitenumfang	12
ISSN	0301-4622
DOIs	https://doi.org/10.1016/0301-4622(93)80017-D
Publikationsstatus	Veröffentlicht - 01.05.1993

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Thanks are due to following members of the lnstitut fiir Biochemie: Manfred Dewor for the amino acid analyses, to Jiirgen Stahl for the CD measurements, to Berthold Wroblowski for determining the secondary structural composition of rhC5a on the bases of these results and to Edgar Jacoby for computer graphic distance determinations. Furthermore, we wish to thank Dr. G. Marius Clore for providing the backbone coordinates of the NMR structure of bovine C5a. This work was supported by a grant from the Bun-desminister fiir Forschung und Technologie der Bundesrepublik Deutschland (Fbrderkenn-zeichen 01 VM 8901/4X

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title = "Tryptophan mutants of human C5a anaphylatoxin: A fluorescence anisotropy decay and energy transfer study",

abstract = "Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 ± 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.",

author = "M. Federwisch and A. Wollmer and M. Emde and T. St{\"u}hmer and T. Melcher and A. Klos and J. K{\"o}hl and W. Bautsch",

year = "1993",

month = may,

day = "1",

doi = "10.1016/0301-4622(93)80017-D",

language = "English",

volume = "46",

pages = "237--248",

journal = "Biophysical Chemistry",

issn = "0301-4622",

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TY - JOUR

T1 - Tryptophan mutants of human C5a anaphylatoxin: A fluorescence anisotropy decay and energy transfer study

AU - Federwisch, M.

AU - Wollmer, A.

AU - Emde, M.

AU - Stühmer, T.

AU - Melcher, T.

AU - Klos, A.

AU - Köhl, J.

AU - Bautsch, W.

PY - 1993/5/1

Y1 - 1993/5/1

N2 - Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 ± 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.

AB - Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 ± 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.

UR - https://www.scopus.com/pages/publications/0027212582

U2 - 10.1016/0301-4622(93)80017-D

DO - 10.1016/0301-4622(93)80017-D

M3 - Journal articles

C2 - 8343570

AN - SCOPUS:0027212582

SN - 0301-4622

VL - 46

SP - 237

EP - 248

JO - Biophysical Chemistry

JF - Biophysical Chemistry

IS - 3

ER -

Tryptophan mutants of human C5a anaphylatoxin: A fluorescence anisotropy decay and energy transfer study

Abstract

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