Contribution of extracellular Glu residues to the structure and function of bacteriorhodopsin. Presence of specific cation-binding sites.
Single and multiple mutants of extracellular Glu side chains of bacteriorhodopsin were analyzed by acid and calcium titration, differential scanning calorimetry, and thermal difference spectrophotometry. Acid titration spectra show that the second group protonating with Asp(85) is revealed in E204Q in the absence of Cl(-) but is not observed in the triple mutant E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q. The results point to Glu(9) as the second group protonating cooperatively with Asp(85). Comparison of the apparent pK(a) of Asp(85) protonation in water and in the deionized forms and results of calcium titration suggest that cation-binding sites are of low affinity in the multiple Glu mutants. Like for deionized wild type bacteriorhodopsin, differential scanning calorimetry reveals a lack of the pretransition in the multiple mutants, whereas in E9Q it appears at lower temperature and with lower cooperativity. Additionally, at neutral pH the band at 630 nm arising from cation release upon temperature increase is absent for the multiple mutants. Based on these results, we propose the presence of two cation-binding sites in the extracellular region of bacteriorhodopsin having as ligands Glu(9), Glu(194), Glu(204), and water molecules.
Sanz
C
C
Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Barcelona 08193, Spain.
Márquez
M
M
Perálvarez
A
A
Elouatik
S
S
Sepulcre
F
F
Querol
E
E
Lazarova
T
T
Padrós
E
E
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D002412:Cations; D005110:Extracellular Space; D018698:Glutamic Acid / Q000378:metabolism*; D006217:Halobacterium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011489:Protein Denaturation; D013329:Structure-Activity Relationship
D002412:Cations; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D002118:Calcium