A reinvestigation of the secondary structure of functionally active vSGLT, the vibrio sodium/galactose cotransporter.
The bacterial Na(+)/galactose cotransporter vSGLT of Vibrio parahaemolyticus is a member of the sodium:solute symporter family (SSS). Previous studies using electron microscopy have shown that vSGLT is a monomeric protein. Computational and experimental topological analyses have consistently indicated that this protein possesses 14 transmembrane alpha-helices. Our previous study using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) to quantitate secondary structure content had indicated, in contrast, an alpha-helical content of only 35%, too little to be consistent with the 14-span model [le Coutre, J., et al. (2002) Biochemistry 41, 8082-6]. ATR-FTIR had also indicated that upon binding of Na(+) and d-galactose, the alpha-helical content increased to 53%. Here we revisit the vSGLT secondary structural distribution using an alternative approach, ultraviolet circular dichroism spectropolarimetry (CD), which is highly accurate in determining the alpha-helical content of a protein in solution. CD spectra were obtained from actively functional, soluble vSGLT and, as an internal check, from a fusion protein of vSGLT and the beta-barrel green fluorescent protein (GFP). Far-UV CD of vSGLT indicates a predominating 85% alpha-helical content, and an absence of beta-strands. Far-UV CD of the vSGLT-GFP fusion corroborates this profile, indicating an equivalent alpha-helical content, and a beta-strand content consistent with the GFP contribution. No detectable substrate-induced macroscopic changes in secondary structure are apparent in the far UV. In the near UV, increases in positive CD intensity occur in a stepwise manner with added substrates, implying changing environments of aromatic amino acid residues. CD thus confirms the current 14-transmembrane span model of vSGLT and reveals distinct substrate-induced conformational changes. The high percentage of alpha-helical structure found requires, when considered in the context of membrane topology, that nearly a third of the total alpha-helical fraction lies in extramembrane domains, which distinguishes this cotransporter from the unrelated lactose and glycerol 3-phosphate transporters.
Turk
Eric
E
Department of Physiology, David Geffen School of Medicine at UCLA, University of California, 90095-1751, USA. eturk@mednet.ucla.edu
Gasymov
Oktay K
OK
Lanza
Seren
S
Horwitz
Joseph
J
Wright
Ernest M
EM
D002942:Circular Dichroism; D005690:Galactose / Q000737:chemistry / Q000502:physiology; D006021:Glycophorins / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D008823:Micelles; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D012964:Sodium / Q000737:chemistry / Q000502:physiology; D051269:Sodium-Glucose Transporter 1 / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D014736:Vibrio parahaemolyticus / Q000737:chemistry / Q000502:physiology
D006021:Glycophorins; D008823:Micelles; D011993:Recombinant Fusion Proteins; D051269:Sodium-Glucose Transporter 1; D049452:Green Fluorescent Proteins; D012964:Sodium; D005690:Galactose