Contribution of individual tryptophan residues to the fluorescence spectrum of native and denatured forms of human carbonic anhydrase II.
Measurements were made of fluorescence spectra produced by pseudo-wild-type human carbonic anhydrase II and mutants in which the tryptophan residues had been replaced by phenylalanine or cysteine residues. 2D NMR spectra of 15N-labeled proteins indicated that the mutations had essentially no long range effects on structure and that the pertubations of structure in the vicinity of the mutated Trp were small. The individual contributions of the seven tryptophan residues were deduced from measurements on native proteins and on proteins subjected to various denaturing conditions. Trp97 and Trp245 are the major fluorescence emitters in the native state, contributing 52% and 38%, respectively, to the total fluorescence intensity. Comparisons of the fluorescence yield of pseudo-wild-type human carbonic anhydrase II and mutant proteins also indicate net energy transfer from Trp16 to Trp5 and from Trp192 to Trp209. The fluorescence from Trp5 is efficiently quenched by His64. In addition, acrylamide quenching of fluorescence was used to probe the environment of tryptophans in proteins incubated in 0, 1.5, and 5 M guanidine hydrochloride. The results indicate that the part of the native protein that corresponds to beta-strands 3-7 forms a compact core in a molten globule intermediate.
Mårtensson
L G
LG
Department of Biochemistry, Umeå University, Sweden.
Jonasson
P
P
Freskgård
P O
PO
Svensson
M
M
Carlsson
U
U
Jonsson
B H
BH
D002256:Carbonic Anhydrases / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry*
D006146:Guanidines; D014364:Tryptophan; D002256:Carbonic Anhydrases; D019791:Guanidine