We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, -96, -115, and -212 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their Am=,.X varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85 --Asn mutant showed no detectable proton pumping, the Asp-96 -+ Asn and Asp-212 --Glu mutants showed <10% and the Asp-115 -Glu mutant showed -30% ofthe normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.Bacteriorhodopsin (bR), an integral membrane protein, functions as a light-driven proton pump in Halobacterium halobium. The protein traverses the cytoplasmic membrane seven times and contains one molecule of all-trans-retinal linked as a Schiff base to Lys-216 as the chromophore (Fig. 1). In structure-function studies of this protein, we are investigating the following questions. (i) What is the mechanism of vectorial proton translocation? Does it involve proton conduction through the functional groups of certain specific amino acids in the membrane-embedded regions? (it) What is the nature of the interactions between retinal and the protein and how do these interactions change during different stages of the photochemical cycle? (iii) What is the nature of the interactions between the membrane-embedded segments that lead to a specific folding pathway?By recombinant DNA methods we have carried out a variety of amino acid substitutions that were designed to remove specific functional groups (1-4). All of the mutants bound retinal to regenerate bR-like chromophores, and most showed unchanged light-dependent proton pumping. The mutants could be divided into two groups on the basis of their spectral properties. One group showed essentially the native bR absorption spectrum, whereas the second group showed varying but significant spectral shifts from the native bR spectrum. The only two mutants that showed altered proton pumping were Pro-186 -+ Leu and Tyr-185 --Phe.With the aim of identifying more mutations that affect proton pumping, we have now carried out single substitutions of all the aspartic acid residues (Fig. 1) (it) the mutants Asp-115 -Glu and Asp-212 --Asn showed reduced (35 and 15%, respectively) pumping whereas Asp-212 --Ala was unstable to light and showed no pumping; and (ii,) substitution of Asp-36, -38, -102, -104, and -115 by asparagine residues did not affect proton pumping.We document these findings and discuss their relevance to the mechanism of proton translocation as well ...

The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.

Thrombin appears to activate platelets by a novel mechanism that involves the cleavage of its receptor, and it has been proposed that the newly generated N-terminal region of the receptor then acts as a tethered ligand [Vu, T. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. Peptides with sequences corresponding to those of the tethered ligand are capable of activating the receptor. In the present study, groups within this tethered ligand peptide that are important for activation of the receptor have been identified by synthesizing a series of peptides. A 14-residue peptide based on the tethered ligand stimulated the aggregation of gel-filtered platelets with an EC50 of 7 microM, and a concentration of 10 microM was the minimum concentration necessary to yield a full aggregation response in platelet-rich plasma. Truncation of the peptide from the C-terminus to nine residues did not markedly affect the response to the peptide. Shorter peptides of five, six, and eight amino acids retained their agonist activity, but the minimal concentration necessary to achieve a full aggregation response in platelet-rich plasma was 2-5-fold higher. Side chains within the tethered ligand peptide that are important for receptor activation were identified by synthesizing a series of peptides in which residues were sequentially replaced by alanine. The results indicated that the side chains of phenylalanine, leucine, and arginine in positions 2, 4, and 5, respectively, are essential for full activity. Most notably, substitution of phenylalanine in the second position resulted in complete loss of agonist activity at concentrations up to 800 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

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