T Marti

Time-resolved surface charge change on the cytoplasmic side of bacteriorhodopsin

The pH‐sensitive dye 5‐iodoacetamidofluorescein was covalently bound to a single cysteine residue introduced by site‐directed mutagenesis in position 101 on the cytoplasmic surface or in position 130 on the extracellular surface of the proton pump bacteriorhodopsin. Using time‐resolved absorption spectroscopy at 495 nm a transient increase was observed in the apparent pK of the dye attached at residue 101. At pH 7.3 the rise and decay times of this pK‐change (∼2 ms and ∼60 ms) correlate well with decay times observed for the M and O intermediates and with the proton uptake time. Interpreting the pK‐increase of +0.18 pH‐unit in terms of a transiently more negative surface charge density, we calculate a change of −0.80 elementary charge per bacteriorhodopsin at the cytoplasmic surface. It is likely that this charge change is due to the transient deprotonation of aspartate‐96. With the label in position 130 on the extracellular surface no transient pK‐shift was detected.

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

Abstract— 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 ofPro–50,Pro–91 andPro–186 on the deprotonation and reprotonation kinetics of the Schiffbase (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 thatPro–50 andPro–91, as well asPro–186, are important for the proton‐pumping function of bR.

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr−) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results or NMR, UV Raman as well as electron cryo‐microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp‐182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbation of the lowest energy absorption of Trp‐182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.

Site-directed isotope labeling of membrane proteins: A new tool for spectroscopists

Publisher Summary This chapter introduces a method for assigning bands in Fourier transform infrared (FTIR)-difference spectra based on a technique termed as site-directed isotope labeling (SDIL). The key element in SDIL is the use of a suppressor tRNA aminoacylated with an isotopically labeled amino acid. This tRNA is targeted to insert the isotopic amino acid at the proper position in the nascent protein by using an amber codon at the corresponding position in the gene. Cell-free synthesis (in vitro translation) and exogenous addition of the aminoacylated suppressor tRNA prevent aminoacylation of non-suppressor tRNAs with the isotopic amino acid, similar to the approach used for site-directed non-native amino acid replacement. The integral membrane protein bacteriorhodopsin (bR) was chosen as a model system for demonstrating the application of SDIL-FTIR. Studies on bacteriorhodopsin demonstrate that the FTIR-SDIL approach can probe the local environment and structural changes of specific residues and backbone carbonyl groups in a protein.

The Identification and Significance of Substructural Domains

Several quite independent lines of evidence provide increasingly forceful arguments for the importance of substructural domains in the biological function of proteins. Models based on these arguments describe independently folded domains with unique binding capacities. Interactions between these domains offer opportunities for synergism and regulation.