Nonpolar environment of tryptophans in erythrocyte water channel CHIP28 determined by fluorescence quenching.
CHIP28 is an abundant water-transporting protein in erythrocytes, kidney proximal tubule, and other fluid-transporting tissues. To determine the environment of the four tryptophans in CHIP28, fluorescence spectra and quenching by polar and nonpolar compounds were measured in stripped human erythrocyte membranes containing CHIP28 and in proteoliposomes reconstituted with purified CHIP28; comparative studies were performed in membranes containing MIP26. Functional analysis showed that CHIP28 water permeability was not affected by the polar quenchers iodide and acrylamide nor the nonpolar n-anthroyloxy fatty acids (n-AF). The emission maximum of CHIP28 tryptophan fluorescence was at 324 +/- 2 nm and did not change with the addition of quenchers; the maximum for MIP26 was at 335 +/- 5 nm. There was weak quenching of CHIP28 tryptophan fluorescence by the polar compounds iodide and acrylamide, with Stern-Volmer constants of 0.13 and 0.71 M-1, respectively. HgCl2 inhibited water permeability by > 95% at 50 microM and quenched CHIP28 fluorescence reversibly by up to 70% with a biphasic concentration dependence; quenching by HgCl2 and acrylamide was not additive. The membrane-associated n-AF probes quenched CHIP28 fluorescence by up to 80% with the greatest quenching for n = 2 and 12; addition of HgCl2 or acrylamide after n-AF caused a small, anthroyloxy-position-dependent increase in quenching which was greatest at n = 6. These studies indicate that the tryptophans in CHIP28 are in a nonpolar, membrane-associated environment. Mathematical modeling of the n-AF results suggests that the tryptophans are clustered near the surface and center of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
Farinas
J
J
Department of Medicine, University of California, San Francisco 94143-0532.
Van Hoek
A N
AN
Shi
L B
LB
Erickson
C
C
Verkman
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001483:Base Sequence; D001789:Blood Group Antigens; D002463:Cell Membrane Permeability; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007672:Kidney Cortex / Q000378:metabolism; D008081:Liposomes; D008565:Membrane Proteins / Q000097:blood / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011510:Proteolipids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan; D014981:Xenopus
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D017931:DNA Primers; D008081:Liposomes; D008565:Membrane Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D051398:Aquaporin 1; D014364:Tryptophan