Replacement of the Arg residue at position 82 in bacteriorhodopsin by Gln or Ala was previously shown to slow the rate of proton release and raise the pK of Asp 85, indicating that R82 is involved both in the proton release reaction and in stabilizing the purple form of the chromophore. We now find that guanidinium chloride lowers the pK of D85, as monitored by the shift of the 587-nm absorbance maximum to 570 nm (blue to purple transition) and increased yield of photointermediate M. The absorbance shift follows a simple binding curve, with an apparent dissociation constant of 20 mM. When membrane surface charge is taken into account, an intrinsic dissociation constant of 0.3 M fits the data over a range of 0.2-1.0 M cation concentration (Na+ plus guanidinium) and pH 5.4-6.7. A chloride counterion is not involved in the observed spectral changes, as chloride up to 0.2 M has little effect on the R82Q chromophore at pH 6, whereas guanidinium sulfate has a similar effect to guanidinium chloride. Furthermore, guanidinium does not affect the chromophore of the double mutant R82Q/D85N. Taken together, these observations suggest that guanidinium binds to a specific site near D85 and restores the purple chromophore. Surprisingly, guanidinium does not restore rapid proton release in the photocycle of R82Q. This result suggests either that guanidinium dissociates during the pump cycle or that it binds with a different hydrogen-bonding geometry than the Arg side chain of the wild type.
Peptides are short chain of amino acids linked by peptide bonds. They are widely used as effective and biocompatible active ingredients in cosmetic industry. In this study, we developed novel peptide mixture and identified its anti-pigmentation effect on melanocytes and keratinocytes. Our results revealed that peptide mixture inhibited melanosome biogenesis through the regulation of microphthalmia-associated transcription factor, a key factor of melanogenesis in melanocytes. And we observed that peptide mixture inhibited melanosome uptake through the reduction of protease-activated receptor 2, a phagocytosis-related receptor in keratinocytes. Furthermore, peptide mixture activated autophagy system resulting in degradation of transferred melanosomes in keratinocytes. The anti-pigmentation effect of multi-targeting peptide mixture was assessed in a human skin equivalent model (MelanoDerm). Melanin contents in epidermal layer were significantly decreased by topical treatment of peptide mixture, suggesting that it can be applied as a novel cosmetics material having a whitening function.
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