Conformational changes in human red cell membrane proteins induced by sugar binding

Abstract

We have previously shown that the human red cell glucose transport protein and the anion exchange protein, band 3, are in close enough contact that information can be transmitted from the glucose transport protein to band 3. The present experiments were designed to show whether information could be transferred in the reverse direction, using changes in tryptophan fluorescence to report on the conformation of the glucose transport protein. To see whether tryptophan fluorescence changes could be attributed to the glucose transport protein, we based our experiments on procedures used by Helgerson and Carruthers [Helgerson, A. L., Carruthers, A., (1987) J. Biol. Chem. 262:5464-5475] to displace cytochalasin B (CB), the specific D-glucose transport inhibitor, from its binding site on the inside face of the glucose transport protein, and we showed that these procedures modified tryptophan fluorescence. Addition of 75 mM maltose, a nontransportable disaccharide which also displaces CB, caused a time-dependent biphasic enhancement of tryptophan fluorescence in fresh red cells, which was modulated by the specific anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate). In a study of nine additional disaccharides, we found that both biphasic kinetics and DBDS effects depended upon specific disaccharide conformation, indicating that these two effects could be attributed to a site sensitive to sugar conformation. Long term (800 sec) experiments revealed that maltose binding (+/- DBDS) caused a sustained damped anharmonic oscillation extending over the entire 800 sec observation period. Mathematical analysis of the temperature dependence of these oscillations showed that 2 microM DBDS increased the damping term activation energy, 9.5 +/- 2.8 kcal mol-1 deg-1, by a factor of four to 39.7 +/- 5.1 kcal mol-1 deg-1, providing strong support for the view that signalling between the glucose transport protein and band 3 goes in both directions.

Genetic Engineering of an Allosterically Based Glucose Indicator Protein for Continuous Glucose Monitoring by Fluorescence Resonance Energy Transfer

“…Most biosensors use proteins, which provide the desired analyte specificity, but often are not appropriate for noninvasive detection because they lack an intrinsic signal transduction mechanism . A variety of glucose-binding proteins have been isolated and well characterized. − These proteins are highly specific for glucose binding, but do not provide any optical signal in the visible region when glucose binding occurs. Intensive efforts have been made to introduce signal transduction functions into these glucose-binding proteins for noninvasive optical glucose biosensors. , …”

Glucose Transporter Function Is Controlled by Transporter Oligomeric Structure. A Single, Intramolecular Disulfide Promotes GLUT1 Tetramerization

“…Several independent lines of evidence demonstrate that in situ, the glucose transporter is a GLUT1 homotetramer Jarvis et al, 1986;Jung et al, 1980;Pessino et al, 1991;Zoccoli et al, 1978) and that the kinetics of transport differ fundamentally from those expected of an E1-E2 carrier. Rather than exposing import and export sites sequentially, the erythrocyte sugar transporter appears to expose import and export sites simultaneously (Baker & Naftalin, 1979;Carruthers, 1986aCarruthers, , 1991; Chin et al, 1992; Hebert & Carruthers, 1991& Carruthers, , 1992Helgerson & Carruthers, 1987;Janoshazi et al" 1991;Janoshazi & Solomon, 1993;Naftalin, 1988; Naftalin & Rist, 1994). One hypothesis that accounts for this behavior suggests that subunits of tetrameric GLUT1 behave as EFE2 carriers in isolation but present conformational states with a pseudo-Z)2 symmetry in the parental structure (Hebert & Carruthers, 1992).…”