Secondary structure analysis of purified functional CHIP28 water channels by CD and FTIR spectroscopy.
The integral membrane protein CHIP28 is an important water channel in erythrocytes and kidney tubule epithelia and is a member of a family of channel/pore proteins including the lens protein MIP26. The purposes of this study were to purify functional, delipidated CHIP28 to homogeneity and to determine secondary structure by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). CHIP28 was initially purified and delipidated by anion-exchange chromatography following solubilization of N-lauroylsarcosine-stripped erythrocyte membranes with beta-octylglucoside (OG); MIP26 was initially purified and delipidated by anion-exchange chromatography following solubilization of urea-stripped bovine lens membranes by monomyristoylphosphatidylcholine. CHIP28 (glycosylated and nonglycosylated) and MIP26 were purified further by high-performance size-exclusion chromatography, eluting in OG as apparent dimers and tetramers, respectively. Proteoliposomes reconstituted with purified CHIP28 were highly water-permeable, with an osmotic water permeability Pf of 0.04 cm/s at 10 degrees C that was inhibited by 0.1 mM HgCl2. Proteoliposomes reconstituted with MIP26 had a low Pf of 0.005 cm/s. CD spectra of CHIP28 in OG or in reconstituted proteoliposomes gave a maximum at 193 nm and minima at 208 and 222 nm. Spectral decomposition using protein basis spectra gave 40 +/- 5% alpha-helix and 43 +/- 3% beta-sheet and -turn. HgCl2 did not affect the CD spectrum of CHIP28. Attenuated total reflectance FTIR of air-dried, membrane-associated CHIP28 gave 38 +/- 5% alpha-helix and 40 +/- 4% beta-sheet and -turn by spectral decomposition of the amide I resonance. For comparison, CD of MIP26 in OG gave 49 +/- 7% alpha-helix and 32 +/- 12% beta-sheet and -turn; FTIR gave 32 +/- 8% alpha-helix and 45 +/- 6% beta-sheet and -turn. Analysis of CHIP28 and MIP26 sequence data by the generalized hydropathy method of Jähnig [Jähnig, F. (1990) Trends Biochem. Sci. 15, 93-95] predicted 39-47% alpha-helix and 15-20% beta-structures. These results establish procedures to obtain large quantities of pure CHIP28 and MIP26 in functional forms and provide evidence for multiple membrane-spanning alpha-helices or mixed alpha/beta-domains.
Van Hoek
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Department of Medicine, University of California, San Francisco 94143.
Wiener
M
M
Bicknese
S
S
Miercke
L
L
Biwersi
J
J
Verkman
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AS
D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D010539:Permeability; D010750:Phosphoproteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010750:Phosphoproteins; C030328:aquaporin 0; D051398:Aquaporin 1