Consequences of amino acid insertions and/or deletions in transmembrane helix C of bacteriorhodopsin (membrane protein/proton transport/protein structure/mutagenesis/kinetic spectroscopy)

Six bacterioopsin mutants containing either single amino acid deletions (AA84, AL87), insertions (V85A, V88A), or both deletions and insertions (AA84/V88A, V85A/AL87) within the first two turns of transmembrane helix C, starting from the extracellular side, have been prepared. The mutant apoproteins refold in phospholipid/detergent mi- celles and display secondary structures similar to that of the wild type. However, the mutants V88A and AA84/V88A do not form a chromophore with retinal. The regenerated chro- mophore of V85A displays absorption maxima and retinal isomer compositions in the dark- and light-adapted states similar to those of the wild type. In AA84, AL87, and V85A/AL87 these chromophore properties are altered, and the structures are less stable than that of the wild type, as shown by an enhanced rate of reaction with hydroxylamine in the dark, an increased pKa of the denaturation at acidic pH, and a decreased pKa of Schiff base deprotonation. Proton translo- cation is abolished in the AA84 and V85A/AL87 mutants, whereas in V85A and AL87 the activity is reduced to about 25 % of the wild-type value at pH 6. The overall properties of the