Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca[sup 2+] binding to deionized bacteriorhodopsin
- Univ. of California, Los Angeles (United States)
- Univ. of California, Irvine (United States)
- Wayne State Univ., School of Medicine Detroit, MI (United States)
Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca[sup 2+]-binding sites. In our efforts to find the location of these Ca[sup 2+] sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca[sup 2+] ions in Ca[sup 2+]-regenerated bR were examined. The important results are as follows: (i) The removal of Ca[sup 2+] from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Try-185, was replaced by a non-H-bonding amino acid (Phe), (ii) Similar to the observation in wild-type bR, the binding of Ca[sup 2+] to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca[sup 2+]. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the exten of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Try-185 was replaced with Phe(Y185F). The above results suggest that the two Ca[sup 2+] ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. 37 refs., 3 figs., 2 tabs.
- DOE Contract Number:
- FG03-86ER13525
- OSTI ID:
- 6653619
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 90:4; ISSN PNASA6; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METALS
BACTERIA
BARYONS
BIOCHEMISTRY
CALCIUM
CATIONS
CELL CONSTITUENTS
CELL MEMBRANES
CHARGED PARTICLES
CHEMICAL BONDS
CHEMISTRY
COLOR
ELEMENTARY PARTICLES
ELEMENTS
FERMIONS
GENE RECOMBINATION
HADRONS
IONS
MEMBRANES
METALS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEONS
OPTICAL PROPERTIES
ORGANIC COMPOUNDS
ORGANOLEPTIC PROPERTIES
PHYSICAL PROPERTIES
PIGMENTS
PROTEINS
PROTONS
PUMPING
RESIDUES
RHODOPSIN