Effects of individual genetic substitutions of arginine residues on the deprotonation and reprotonation kinetics of the Schiff base during the bacteriorhodopsin photocycle

Lin, Gloria C.; El‐Sayed, Mostafa A.; Marti, Thomas; Stern, Lawrence J.; Mogi, Tatsushi; Khorana, H G

doi:10.1016/s0006-3495(91)82040-9

Cited by 15 publications

(12 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our strategy was to solve the first half of the cycle in the way it was done previously, 3 The values for M decay in wild-type and R227Q are 1.5 and 6.3 ms, respectively, and the tz values are 26 and 24 ms (with amplitude ratios of 66:34 in wild-type and 22:78 in R227Q). This is the opposite of what was reported for the photocycle of the R227Q mutant when expressed in Escherichia coli (Lin et al, 1991). In that system, t\ was unchanged from wild-type, but T2 became 5 times slower.…”

Section: Resultscontrasting

confidence: 87%

Pathway of proton uptake in the bacteriorhodopsin photocycle

Zimányi¹,

Cao²,

Needleman³

et al. 1993

Biochemistry

View full text Add to dashboard Cite

The time courses of chromophore reactions and proton uptake in the second half of the photocycle of the proton pump bacteriorhodopsin (BR) were examined. At pH > 8.5, the kinetics are simplified by the fact that only the M and N intermediates accumulate. The relaxation kinetics after perturbation of M with a second, blue flash confirm that M<==>N equilibration is the only significant cause of the biphasic M decay. With this feature, the analysis of time-resolved difference spectra yields a scheme which contains two sequential N states connected by a nearly unidirectional reaction. The proton uptake from the bulk, as measured with the pH-indicator dye pyranine, occurs during the decay of the first N rather than the recovery of BR. The results thus suggest the model M2(-1)<==>N(-1) + H+ (from the bulk)<==>N(0)-->BR, where the superscripts indicate the protonation state of the protein relative to BR. M2(-1)-->N(-1) is reprotonation of the Schiff base from D96, N(-1) + H+ (from the bulk)-->N(0) is uptake of proton from the cytoplasmic side, and N(0)-->BR represents 13-cis to all-trans reisomerization of the retinal and other relaxations which regenerate the initial state. R227, a residue near D96, affects the deprotonation of D96 more than the subsequent proton uptake. According to the changed [M2(-1)]/[N(-1)] equilibrium in the R227Q protein, interaction between R227 and D96 is responsible for about 1 pH unit of the decrease in the pKa of D96 during the reprotonation of the Schiff base. According to the pH dependencies of the rate constants in the N(-1)<==>N(0) equilibrium in wild-type and R227Q, interaction with R227 lowers the pKa for proton uptake from the bulk by 0.5 pH unit, to 11. We conclude from the proton uptake kinetics that at physiological pH free energy is converted to proton electrochemical potential in this pump not only as protons are released on the extracellular side [Zimányi, L., Váró. G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] but also as protons are taken up on the cytoplasmic side.

show abstract

Section: Resultscontrasting

confidence: 87%

Pathway of proton uptake in the bacteriorhodopsin photocycle

Zimányi¹,

Cao²,

Needleman³

et al. 1993

Biochemistry

View full text Add to dashboard Cite

show abstract

“…1 and in comparison with the kinetics data obtained by Lin et a[. 33 and Wu et al" for other mutants, the significance in the change of the SB deprotonation rate caused by the mutation at site Pro-SO and Pro-9 1 are compatible to, if not larger than those caused by the replacements of many amino acid residue^^'. '^ that are assigned to be in the proton channel.…”

Section: Deprotonation (M-formation) and Reprotonation (M-decay) Kinesupporting

confidence: 60%

EFFECTS OF MUTAGENETIC SUBSTITUTION OF PROLINES ON THE RATE OF DEPROTONATION and REPROTONATION OF THE SCHIFF BASE DURING THE PHOTOCYCLE OF BACTERIORHODOPSIN

et al. 1993

Photochem & Photobiology

View full text Add to dashboard Cite

Membrane-buried proline residues are found in many transport proteins. To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions of Pro-50, Pro-91 and Pro-186 on the deprotonation and reprotonation kinetics of the Schiff base (SB) were determined by flash photolysis. The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli). The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant. These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate. Our results indicate that Pro-50 and Pro-91, as well as Pro-186, are important for the proton-pumping function of bR.

show abstract

“…More evidence for such a network was recently obtained by FTIR spectroscopy (le Coutre et al, 1995). The network could explain the interaction of Arg227 with Asp96 inferred from the slowed photocycle observed in the Arg227 to Gln mutant (Stern & Khorana, 1989;Lin et al, 1991) and the decreased pK a of Asp96 in M (Zimányi et al, 1993). The suggested salt bridge between Arg227 and Asp96 (Stern & Khorana, 1989) seems unlikely in view of the distance (about 9 Å ) between the side-chains of these two residues in the refined model.…”

Section: Cavities Water Molecules and H-bonding Networkmentioning

confidence: 90%