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Binding of calcium ions to bacteriorhodopsin
G Váró1, L S Brown, R Needleman
1Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Biophysical Journal
|June 4, 1999
Summary
Calcium ions (Ca2+) do not bind directly to bacteriorhodopsin but indirectly raise surface pH, causing Asp85 deprotonation. This pH effect explains complex spectral changes observed in bacteriorhodopsin and its mutants.
Area of Science:
- Biochemistry
- Biophysics
- Membrane Protein Dynamics
Background:
- Bacteriorhodopsin undergoes spectral shifts upon cation addition, linked to Asp85 deprotonation.
- The mechanism of Ca2+ influence on Asp85 protonation is debated: direct binding vs. indirect surface pH changes.
Purpose of the Study:
- To investigate whether Ca2+ binds directly to bacteriorhodopsin or affects it indirectly via surface pH.
- To elucidate the role of surface pH in cation-induced spectral changes of bacteriorhodopsin.
Main Methods:
- Engineered cysteine residues with covalently linked eosin to monitor surface pH.
- Controlled addition of Ca2+ to deionized bacteriorhodopsin samples.
- Spectroscopic analysis of color shifts and protonation states.
Main Results:
- Ca2+ addition increased surface pH equally on both protein surfaces, ruling out specific binding sites.
- Ca2+ binding to anionic lipids was identified as the cause of surface pH increase.
- Asp85 deprotonation occurred only when surface pH approached its pKa, revealing a nonlinear relationship.
Conclusions:
- Ca2+ indirectly influences bacteriorhodopsin by increasing surface pH, not by direct binding.
- The observed spectral titration curves are explained by the nonlinear Ca2+ binding-surface pH-Asp85 deprotonation relationship.
- This mechanism is consistent across wild-type and mutant bacteriorhodopsin variants.
Keywords:
Non-programmatic