The role of small intraprotein cavities in the catalytic cycle of bacteriorhodopsin

The last phase of the proton transfer cycle of bacteriorhodopsin calls for a passage of a proton from D38 to D96. This reaction utilizes a narrow shaft approximately 10-A long that connects the two carboxylates that cross through a very hydrophobic domain. As the shaft is too narrow to be permanently hydrated, there are two alternatives for the proton propagation into the channel. The proton may propagate through the shaft without solvation at the expense of a high electrostatic barrier; alternatively, the shaft will expand to accommodate some water molecules, thus lowering the Born energy for the insertion of the charge into the protein (B. Sch?tzler, N. A. Dencher, J. Tittor, D. Oesterhelt, S. Yaniv-Checover, E. Nachliel, and G. Gutman, 2003, BIOPHYS: J. 84:671-686). A comparative study of nine published crystal-structures of bacteriorhodopsin identified, next to the shaft, microcavities in the protein whose position and surrounding atoms are common to the reported structures. Some of the cavities either shrink or expand during the photocycle. It is argued that the plasticity of the cavities provides a working space needed for the transient solvation of the shaft, thus reducing the activation energy necessary for the solvation of the shaft. This suggestion is corroborated by the recent observations of Klink et al. (B. U. Klink, R. Winter, M. Engelhard, and I. Chizhov, 2002, BIOPHYS: J. 83:3490-3498) that the late phases of the photocycle (tau >/=1 ms) are strongly inhibited by external pressure.     

Dynamical heterogeneity of specific amino acids in bacteriorhodopsin

Components of biological macromolecules, complexes and membranes are animated by motions occurring over a wide range of time and length scales, the synergy of which is at the basis of biological activity. Understanding biological function thus requires a detailed analysis of the underlying dynamical heterogeneity. Neutron scattering, using specific isotope labeling, and molecular dynamics simulations were combined in order to study the dynamics of specific amino acid types in bacteriorhodopsin within the purple membrane (PM) of Halobacterium salinarum. Motions of leucine, isoleucine and tyrosine residues on the pico- to nanosecond time scale were examined separately as a function of temperature from 20 to 300 K. The dynamics of the three residue types displayed different temperature dependence: isoleucine residues have larger displacements compared to the global PM above 120 K; leucine residues have displacements similar to that of PM in the entire temperature range studied; and tyrosine residues have displacements smaller than that of the average membrane in an intermediate temperature range. Experimental features were mostly well reproduced by molecular dynamics simulations performed at five temperatures, which allowed the dynamical characterisation of the amino acids under study as a function of local environment. The resulting dynamical map of bacteriorhodopsin revealed that movements of a specific residue are determined by both its environment and its residue type.     

Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin

Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.     

Protein degradation in mammary gland explants evidence for limited heterogeneity of protein degradation rates

Mammary gland explants in organ culture were subjected to hormonal manipulation, and rates of protein degradation during 1 and 2 day periods were measured by a double-isotope method. Isotope ratios for protein subunits in subcellular fractions were measured after resolution by two-dimensional polyacrylamide gel electrophoresis. Frequency distribution analysis shows that the isotope ratios for each fraction are coupled predominantly in exponential distributions corresponding to populations of protein subunits with different mean degradation rates. The result also suggest that heterogeneity of protein degradation rates within each population is limited. There is no consistent correlation of degradation rate with protein isoelectric point or subunit molecular weight either overall or within any population of degradation rates. Therefore, the similarity of protein degradation rates within each population is clearly not related to these molecular properties of the proteins.     

Huntington disease: no evidence for locus heterogeneity

A total of 63 families with Huntington disease (HD) were examined for linkage between HD and G8 (D4S10). The families included 57 Caucasian, four Black American, and two Japanese. The combined maximum lod score was 87.69 at theta = 0.04 (99% confidence interval 0.018-0.071). The maximum frequency of recombination was 0.03 in males and 0.05 in females. Fifty-seven families gave positive lod scores; five small families gave mildly negative lod scores. The maximum likelihood estimate of alpha, the proportion of linked loci, was 1.0 with a lower 99% confidence interval of 0.88. These data suggest that there is only one HD locus, although a second rare locus cannot be ruled out.     

CD spectrum of bacteriorhodopsin: Best evidence against exciton model

We summarize the predictions of the exciton model that was originally proposed to explain the observed biphasic band shape of its CD spectrum in the visible region of bacteriorhodopsin (bR). It is shown that to reconcile these predictions with the observed results on the linear dichroism, the retinal isomerization time and, the retinal-retinal distance, the biphasic nature of the observed CD spectrum of bR becomes itself an evidence against the exciton model because of the uncertainty principle.

Reduced bR (RbR), which retains its hexagonal structure, shows a monophasic CD spectrum with relatively small rotational strength as compared to bR. This is shown to disagree with predictions made by the exciton model. The results could best be explained in terms of retinal-protein heterogeneity leading to two or more types of bR in which their retinals suffer opposite sense of intramolecular rotational distortion along their retinal long axis. Such a retinal-protein heterogeneity disappears in reduced bR which is known to have a planar (nondistorted) retinal conjugated system, resulting in a monophasic CD with reduced rotational strength, as observed.

     

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis

Summary Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated³H leucine from the digested membranes.During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.     

HLA-DR typing in coeliac disease: evidence for genetic heterogeneity.

Sixty nine propositi from a family study of coeliac disease were typed for HLA-DR antigens. Sixty three (91%) were found to carry the antigen DR3, which was a significantly greater proportion (p = 9.6 X 10(-24] than among the 168 controls (26%). Concurrently 42 children with the disease were DR typed. Not only was the frequency of DR3 significantly increased in these patients (86% versus 26% in controls; p = 3.1 X 10(-12] but so also was the frequency of DR7 (patients 60%, controls 29%; p = 5.8 X 10(-4]. When those propositi whose coeliac disease presented before the age of 20 were combined with the childhood coeliac group and a comparison made between these patients and the remainder of the propositi, all of whom presented when they were older than 20, the childhood onset group had a significant excess of DR7 (p = 2.2 X 10(-3] and a significant deficiency of DR2 (p = 3.5 X 10(-3]. These findings indicate that childhood coeliac disease and adult coeliac disease are genetically heterogeneous.     

Location and orientation of the chromophore in bacteriorhodopsin: Analysis by fluorescence energy transfer

The spatial location and orientation of the retinal chromophore in bacteriorhodopsin were estimated from a fluorescence energy transfer study. The energy donor used in this study was a fluorescent retinal derivative, which was obtained by partial reduction of the purple membrane with sodium borohydride, and the energy acceptor was the native chromophore remaining in the same membrane. Since bacteriorhodopsin forms a two-dimensional crystal with P₃ symmetry in the purple membrane, and the membrane structure is maintained after the reduction, the rate of energy transfer from a donor to any acceptor existing in the same membrane can be calculated as a function of the location and orientation of the chromophores in the unit cell. Quantitative analyses of the fluorescence decay curve and the quantum yield, with various extents of reduction, enabled us to determine the most probable location and orientation. The result suggested that the chromophore was situated near the centre of the protein in such an orientation that the dipole-dipole interaction with neighbouring chromophores was close to minimum.     

Physical and functional heterogeneity in TYMV RNA: evidence for the existence of an independent messenger coding for coat protein.

Turnip yellow mosaic virus RNA can be separated into two distinct components of 2 times 10(6) and 300 000 daltons molecular weight after moderate heat treatment in the presence of SDS or EDTA. The two species cannot have arisen by accidental in vitro degradation of a larger RNA, as they both possess capped 5' ends. Analysis of the newly synthesized proteins resulting from translation of each RNA by a wheat germ extract shows that the 300 000 molecular weight RNA can be translated very efficiently into coat protein. When translated in vitro the longer RNA gave a series of high molecular weight polypeptides but only very small amounts of a polypeptide having about the same mass as the coat protein. Thus our results suggest that the small RNA is the functional messenger for coat protein synthesis in infected cells.     

Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP1 synaptic vesicles banding at 1.055 g/mL actively transported [3H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [3H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP1 vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle originally described in intact Torpedo marmorata electric organ [Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 715-730]. The properties of the receptor for vesamicol were studied by measuring binding of [3H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP1 vesicles the VP2 vesicles had a ratio of [3H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. Based on an antibody standardization, in a typical preparation the VP1 vesicles contained 237 +/- 15 pmol of receptor/mg of protein whereas VP2 vesicles contained 102 +/- 3 pmol of receptor/mg of protein, and VP2 vesicles transported AcCh 2-3-fold more actively than VP1 vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR probes of vectoriality in the proton-motive photocycle of bacteriorhodopsin: evidence for an 'electrostatic steering' mechanism

In recent years, significant progress has been made in elucidating the structure of bacteriorhodopsin. However, the molecular mechanism by which vectorial proton motion is enforced remains unknown. Given the advantages of a protonated Schiff base for both photoisomerization and thermal reisomerization of the chromophore, a five-state proton pump can be rationalized in which the switch in the connectivity of the Schiff base between the two sides of the membrane is decoupled from double bond isomerization. This decoupling requires tight control of the Schiff base until it is deprotonated and decisive release after it is deprotonated. NMR evidence has been obtained for both the tight control and the decisive release: strain develops in the chromophore in the first half of the photocycle and disappears after deprotonation. The strain is associated with a strong interaction between the Schiff base and its counterion, an interaction that is broken when the Schiff base deprotonates. Thus the counterion appears to play a critical role in energy transduction, controlling the Schiff base in the first half of the photocycle by 'electrostatic steering'. NMR also detects other events during the photocycle, but it is argued that these are secondary to the central mechanism.     

Further evidence for the heterogeneity of serum albumin

Albumin samples from three species (avian, bovine and human) were electrophoresed on gradient polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS-PAGE). The resulting electrophoregram from each sample of serum albumin investigated showed multiple protein bands of a wide range of molecular weights. All seven samples of human serum albumin were found, using gel immunodiffusion, to be contaminated with other proteins. All but one sample was contaminated with proteins such as haptoglobin, alpha 1-glycoprotein, alpha 1-trypsin inhibitor, and prealbumin. This contamination accounts for part of the heterogeneity of these samples. Immunoblots, where the proteins were transferred to nitrocellulose and incubated with antisera, gave a better demonstration of the heterogeneity than Coomassie Blue staining and the immunoblotting procedure appeared to be more sensitive than the gel immunodiffusion technique. The heterogeneity of serum albumin demonstrated by the former technique included that of the monomer which was shown to be contaminated with antithrombin III. The commercial samples of human serum albumin, claimed as pure, were found to vary greatly in their tryptophan content, which also indicated heterogeneity. Heat treatment of human serum albumin with 1% SDS, followed by chromatography on agarose, removed the protein contaminants and with it the tryptophan. The presence of tryptophan in human serum albumin, therefore, indicated the presence of impurities.     

Excited-state dynamics of bacteriorhodopsin probed by broadband femtosecond fluorescence spectroscopy

The impact of varying excitation densities (approximately 0.3 to approximately 40 photons per molecule) on the ultrafast fluorescence dynamics of bacteriorhodopsin has been studied in a wide spectral range (630-900 nm). For low excitation densities, the fluorescence dynamics can be approximated biexponentially with time constants of <0.15 and approximately 0.45 ps. The spectrum associated with the fastest time constant peaks at 650 nm, while the 0.45 ps component is most prominent at 750 nm. Superimposed on these kinetics is a shift of the fluorescence maximum with time (dynamic Stokes shift). Higher excitation densities alter the time constants and their amplitudes. These changes are assigned to multi-photon absorptions.