Polymorphism of the C3b/C4b receptor (CR1): characterization of a fourth allele

The receptor for C3b/C4b (C3bR or CR1) has an unusual polymorphism in which three codominant alleles determine variants with a large difference in Mr (160,000, 190,000, or 220,000). We found an individual who has, in addition to the common 190,000 Mr molecule, a C3bR whose Mr is 250,000. In this proband and in some members of his family, this novel heterozygous phenotype can be isolated from 125I surface-labeled cells by iC3 or iC4 affinity chromatography or by immunoprecipitation with the use of polyclonal or monoclonal anti-C3bR. Relative to the 190,000 Mr C3bR, E from individuals in this family have 20 to 30% of the total receptor counts in the 250,000 Mr C3bR. However, on C3bR-bearing leukocytes there is a much larger amount of the 250,000 Mr C3bR (approximately 60%) relative to the 190,000 Mr C3bR. Similar to the other three C3bR variants, the Mr is 5,000 greater on polymorphonuclear cells than on E, and treatment of this new C3bR with endoglycosidase F decreases its Mr by approximately 10,000. Therefore, because this variant is inherited and has structural and functional similarities to the other three C3bR, we conclude that this 250,000 Mr CR1 probably represents a fourth allele.     

Proton transfer reactions in native and deionized bacteriorhodopsin upon delipidation and monomerization

We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the efficiency of M formation and the kinetics of M rise and decay depend on the lipids and lattice but, as the lipids are removed, the cation binding is a much less important factor for the proton pumping function. Upon 75% delipidation using 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS), the M formation and decay kinetics are much slower than the native, and the efficiency of M formation is approximately 30%-40% that of the native. Upon monomerization of bR by Trition X-100, the efficiency of M recovers close to that of the native (depending on pH), M formation is approximately 10 times faster, and M decay kinetics are comparable to native at pH 7. The same results on the M intermediate are observed if deionized blue bR (deI bbR) is treated with these detergents (with or without pH buffers present), even though deionized blue bR containing all the lipids has no photocycle. This suggests that the cation(s) has a role in native bR that is different than in delipidated or monomerized bR, even so far as to suggest that the cation(s) becomes unimportant to the function as the lipids are removed.     

Water molecules and hydrogen-bonded networks in bacteriorhodopsin--molecular dynamics simulations of the ground state and the M-intermediate

Protein crystallography provides the structure of a protein, averaged over all elementary cells during data collection time. Thus, it has only a limited access to diffusive processes. This article demonstrates how molecular dynamics simulations can elucidate structure-function relationships in bacteriorhodopsin (bR) involving water molecules. The spatial distribution of water molecules and their corresponding hydrogen-bonded networks inside bR in its ground state (G) and late M intermediate conformations were investigated by molecular dynamics simulations. The simulations reveal a much higher average number of internal water molecules per monomer (28 in the G and 36 in the M) than observed in crystal structures (18 and 22, respectively). We found nine water molecules trapped and 19 diffusive inside the G-monomer, and 13 trapped and 23 diffusive inside the M-monomer. The exchange of a set of diffusive internal water molecules follows an exponential decay with a 1/e time in the order of 340 ps for the G state and 460 ps for the M state. The average residence time of a diffusive water molecule inside the protein is approximately 95 ps for the G state and 110 ps for the M state. We have used the Grotthuss model to describe the possible proton transport through the hydrogen-bonded networks inside the protein, which is built up in the picosecond-to-nanosecond time domains. Comparing the water distribution and hydrogen-bonded networks of the two different states, we suggest possible pathways for proton hopping and water movement inside bR.     

Photovoltage kinetics of the acid-blue and acid-purple forms of bacteriorhodopsin: evidence for no net charge transfer.

Time-resolved photovoltage measurements were performed with the acid-blue (bR605A) and acid-purple (bR565A) forms of bacteriorhodopsin (bR) in the time range from 25 ns to 100 s. The bR605A and bR565A pigments were formed by titration with H2SO4 in the absence and presence of 150 mM KCI, respectively. Qualitatively the kinetics of the charge displacement in these two states are similar and consist of two fast phases in one direction (100 ns bandwidth limited and approximately 1 microsecond) followed by a decay in the opposite direction via one component for bR605A (4.4 +/- 0.6 ms) or two components for bR565A (33 +/- 8 microseconds and 3.6 +/- 0.5 ms). The transient photovoltage signal returns exactly to the initial value after several milliseconds, well before the passive discharge of the electrical measuring system at 2 s. We conclude that no net charge transfer occurs in either bR605A or bR565A. The direction of the fast components is opposite that of net proton translocation in bR at pH 7. So, if the charge that moves back and forth is due to a proton, it moves first in the direction of the cytoplasmic side of the membrane (< 1 microsecond) and returns to its initial position via the 4.4 ms (bR605A) or the 33 microseconds and 3.6 ms (bR565A) decay components. The amplitude of the charge motion in both low pH forms is too large to be due to isomerization alone and is comparable to one of the major components in bR at pH 7.2     

Cation binding sites on the projected structure of bacteriorhodopsin.

Divalent cations are involved in the function of bacteriorhodopsin (bR) as a light-driven proton pump. If cations are removed from purple membranes they become blue. Divalent cations such as Ca2+ or Pb2+ or trivalent ions, can be stoichiometrically titrated back on to these deionized membranes. The color transitions as a function of ion concentration for Ca2+ or Pb2+ are precisely comparable and indicate that approximately three stoichiometric equivalents of cations are required to effect the color transition (Kimura et al., 1984). We found four main partially occupied binding sites for cations at a stoichiometric ratio of 3 Pb2+/bR. We localized the binding sites for Pb2+ using x-ray diffraction of membranes reconstituted with 1, 2, and 3 equivalents of Pb2+ per bR. The site of highest affinity is located on helix 7. At 2 Pb2+/bR, sites on helix 6 and between helix 2 and 3 are occupied. At 3 Pb2+/bR a fourth site above helix 3 is occupied.     

Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy storage in the primary step of the photocycle of bacteriorhodopsin.

A pulsed-dye laser low temperature photocalorimeter is used to study the enthalpy differences between light-adapted bacteriorhodopsin (bR568) and its primary photoproduct (K) at 77 K. A key feature of our experimental method is the use of the laser-induced photostationary state as an internal reference. Analyses of the forward (bR leads to K), reverse (K leads to bR), and mixed (bR in equilibrium K) photoreactions were carried out to measure delta H12 = EK - EbR. All three experiments yielded identical values of delta H12 within experimental error (delta Have12 = 15.8 +/- 2.5 kcal mol-1). Accordingly, the primary event in the photocycle of light-adapted bacteriorhodopsin stores approximately 30% of the absorbed photon energy at the 568-nm absorption maximum. We observe that the quantum yields phi f1(bR leads to K) and phi r2(K leads to bR) add up to unity within experimental error: phi f1 + phi r2 = 1.02 +/- 0.19 for phi f1 in the range 0.28-0.33. A theoretical analysis of energy storage in K suggests that at least one-half of the enthalpy difference between K and bR is associated with charge separation accompanying chromophore isomerization.     

Simulation analysis of the retinal conformational equilibrium in dark-adapted bacteriorhodopsin

In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two conformers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two forms, using molecular mechanics and dynamics calculations. Adiabatic potential energy mapping indicates that whereas the twofold intrinsic torsional potentials of the C13==C14 and C15==N16 double bonds favor a sequential torsional pathway, the protein environment favors a concerted, bicycle-pedal mechanism. Which of these two pathways will actually occur in bR depends on the as yet unknown relative weight of the intrinsic and environmental effects. The free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type protein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within approximately kBT of experiment. In contrast, in isolated retinal the free energy of the all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. The free energy differences are similar to the adiabatic potential energy differences in the various systems examined, consistent with an essentially enthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein-protein interactions. Removing internal water molecules near the Schiff base strongly stabilizes the (13,15)cis form, whereas a double mutation that removes negative charges in the retinal pocket (Asp85 to Ala; Asp212 to Ala) has the opposite effect.     

Factors affecting the absorption maxima of acidic forms of bacteriorhodopsin. A study with artificial pigments.

The absorption maximum (568 nm) of light-adapted bacteriorhodopsin bR568 undergoes reversible changes after acidification. At pH 2.9, the absorption shifts to 605 nm (forming bR605) and it blue shifts to 565 nm, after further acidification to pH approximately 0.5 (forming bR565). Molecular models accounting for such acid-induced changes are relevant to the structure and function of bacteriorhodopsin. In the present study we approached the problem by applying artificial bR pigments based on selectively modified synthetic retinals. This may allow direct identification of the specific regions in the retinal binding site where the above changes in the protein-retinal interactions take place. We investigated the spectroscopic effects of acid in a variety of artificial pigments, including cyaninelike retinals, retinals bearing bulky groups at C4, short polyenes, and retinals in which the beta-ionone ring was substituted by aromatic rings. The results provide direct evidence for the hypothesis that the generation of bR605 is due to changes in polyene-opsin interactions in the vicinity of the Schiff base linkage. The second transition (to bR565) was not observed in artificial pigments bearing major changes in the ring structure of the retinal. Two approaches accounting for this observation are presented. One argues that the generation of bR565 is associated with acid-induced changes in retinal-protein interactions in the vicinity of the retinal ring. The second involves changes in polyene-opsin interactions in the vicinity of the Schiff base linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation of proline residues in bacteriorhodopsin

Proline, noted as a hydrophilic residue with helix-breaking potential, nevertheless occurs widely in putatively alpha-helical transmembrane segments of many transport proteins. Ligand-activated or enzyme-assisted trans/cis isomerization of an X-proline peptide bond (where X = any amino acid)--a dynamic, reversible event which could alter the orientation of a transmembrane alpha-helix--may provide the molecular basis for a protein channel regulatory process. Further elucidation of such a function requires knowledge of the isomeric status of the X-Pro bonds in native conformations of membrane proteins. We have used 13C nuclear magnetic resonance (NMR) spectroscopy to examine the conformation of intramembranous X-Pro peptide bonds in biosynthetically-labelled samples of a model transport protein, bacteriorhodopsin (bR) (purple membrane). Spectra of 13C-Tyr-carbonyl labelled bR (in the solvent system CHCl3:CD3OD (1:1) + 0.1 M LiClO4) first established that all 11 bR Tyr residues were sufficiently mobile for their resonances to be detected and resolved, independent of their domain location within the bR sequence. By taking advantage of the known diagnostic chemical shifts of the isomers of Pro-C gamma carbon resonances, spectra of bR labelled with 13C gamma-Pro were then used to demonstrate that all 11 bR X-Pro peptide bonds--including those within the protein's membrane domain (Pro50, Pro91, Pro186)--are in the trans conformation in resting state bR.     

Weakened coupling of conserved arginine to the proteorhodopsin chromophore and its counterion implies structural differences from bacteriorhodopsin

In wild-type proteorhodopsin (pR), titration of the chromophore's counterion Asp(97) occurs with a pK(a) of 8.2+/-0.1. R94C mutation reduces this slightly to 7.0+/-0.2, irrespective of treatment with ethylguanidinium. This contrasts with the homologous archaeal protein bacteriorhodopsin (bR), where R82C mutation was previously shown to elevate the pK(a) of Asp(85) by approximately 5 units, while reconstitution with ethylguanidinium restores it nearly to the wild-type value of 2.5. We conclude there is much weaker electrostatic coupling between Arg(94) and Asp(97) in the unphotolyzed state of pR, in comparison to Arg(82) and Asp(85) in bR. Therefore, while fast light-driven H(+) release may depend on these two residues in pR as in bR, no tightly conserved pre-photolysis configuration of them is required.     

A computational model of membrane lipid electronic properties in relation to neural signaling

We present a computational model of a transiently-organized neural membrane molecular system with possible information-processing capacity. The model examines field-induced dipole and quadrupole moments and polarizability in monomeric, dimeric, and trimeric ethenes. Polarization of the ethenes is strongly indicated. This result is interpreted as a significant electronic feature of a molecular computing system based on organization of membrane lipids into a transient ( approximately 10(-4) s) crystalline state due to lipid-protein hydrophobic mismatch at the membrane-ion-channel interface. Predictive implications of the model's electronic features are briefly discussed.     

Functionally relevant coupled dynamic profile of bacteriorhodopsin and lipids in purple membranes

The dynamics of bacteriorhodopsin (bR) and the lipid headgroups in oriented purple membranes (PMs) was determined at various temperatures and relative humidity (rh) using solid-state NMR spectroscopy. The 31P NMR spectra of the alpha- and gamma-phosphate groups in methyl phosphatidylglycerophosphate (PGP-Me), which is the major phospholipid in the PM, changed sensitively with hydration levels. Between 253 and 233 K, the signals from a fully hydrated sample became broadened similarly to those of a dry sample at 293 K. The 15N cross polarization (CP) NMR spectral intensities from [15N]Gly bR incorporated into fully hydrated PMs were suppressed in 15N CP NMR spectra at 293 K compared with those of dry membranes but gradually recovered at low temperatures or at lower hydration (75%) levels. The suppression of the NMR signals, which is due to interference with proton decoupling frequency (approximately 45 kHz), coupled with short spin-spin relaxation times (T2) indicates that the loops of bR, in particular, have motional components around this frequency. The motion of the transmembrane alpha-helices in bR was largely affected by the freezing of excess water at low temperatures. While between 253 and 233 K, where a dynamic phase transition-like change was observed in the 31P NMR spectra for the phosphate lipid headgroups, the molecular motion of the loops and the C- and N-termini slowed, suggesting lipid-loop interactions, although protein-protein interactions between stacks cannot be excluded. The results of T2 measurements of dry samples, which do not have proton pumping activity, were similar to those for fully hydrated samples below 213 K where the M-intermediates can be trapped. These results suggest that motions in the 10s micros correlation regime may be functionally important for the photocycle of bR, and protein-lipid interactions are motionally coupled in this dynamic regime.     

Theoretical framework for octopus rhodopsin crystallization

Bacteriorhodopsin (bR) is presently a classical example of membrane protein crystallization. We are comparing the structure of bR with the homology model of octopus rhodopsin (octR), which is similar in topology to bR and as highly ordered in its native membranes as bR in purple membranes. Such comparison provides insights for optimization of present octR experimentation both for crystallization and for application in nanobiotechnology in a manner similar to bR, and possibly even superior in optical computation.     

Theoretical calculation of polarizability isotope effects

We propose a scheme to estimate hydrogen isotope effects on molecular polarizabilities. This approach combines the any-particle molecular orbital method, in which both electrons and H/D nuclei are described as quantum waves, with the auxiliary density perturbation theory, to calculate analytically the polarizability tensor. We assess the performance of method by calculating the polarizability isotope effect for 20 molecules. A good correlation between theoretical and experimental data is found. Further analysis of the results reveals that the change in the polarizability of a X-H bond upon deuteration decreases as the electronegativity of X increases. Our investigation also reveals that the molecular polarizability isotope effect presents an additive character. Therefore, it can be computed by counting the number of deuterated bonds in the molecule.     

Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states.

Solid-state 13C NMR spectra were employed to characterize the protonation state of tyrosine in the light-adapted (bR568) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for [4'-13C]Tyr-labeled bR, regenerated with [14-13C]retinal as an internal marker to identify the photocycle states. The [14-13C]retinal has distinct chemical shifts for bR555, for bR568, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate are easily distinguished on the basis of the chemical shift of the 4'-13C label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, we show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at -80 degrees C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. In addition, after the M samples were briefly warmed (approximately 30 s), no tyrosinate was observed. However, small (1-2 ppm) changes in the structure or dispersion in the Tyr peak were observed in the M state phototrapped by either method. These changes were reversible when the sample was warmed, although on a time scale slower than the relaxation of the retinal back to the bR568 conformer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a perturbation of arginine-82 in the bacteriorhodopsin photocycle from time-resolved infrared spectra

Arginine-82 (R82) of bacteriorhodopsin (bR) has long been recognized as an important residue due to its absolute conservation in the archaeal rhodopsins and the effects of R82 mutations on the photocycle and proton release. However, the nature of interactions between R82 and other residues of the protein has remained difficult to decipher. Recent NMR studies showed that the two terminal nitrogens of R82 experience a highly perturbed asymmetric environment during the M state trapped at cryogenic temperatures [Petkova et al. (1999) Biochemistry 38, 1562-1572]. Although previous low-temperature FT-IR spectra of wild-type and mutant bR samples have demonstrated effects of R82 on vibrations of other amino acid side chains, no bands in these spectra were assignable to vibrations of R82 itself. We have now measured time-resolved FT-IR difference spectra of bR intermediates in the wild-type and R82A proteins, as well as in samples of the R82C mutant with and without thioethylguanidinium attached via a disulfide linkage at the unique cysteine site. Several bands in the bR --> M difference spectrum are attributable to guanidino group vibrations of R82, based on their shift upon isotope substitution of the thioethylguanidinium attached to R82C and on their disappearance in the R82A spectrum. The frequencies and intensities of these IR bands support the NMR-based conclusion that there is a significant perturbation of R82 during the bR photocycle. However, the unusually low frequencies attributable to R82 guandino group vibrations in M, approximately 1640 and approximately 1545 cm(-)(1), would require a reexamination of a previously discarded hypothesis, namely, that the perturbation of R82 involves a change in its ionization state.     

Herpes simplex virus type 1 infection of endothelial, epithelial, and fibroblast cells induces a receptor for C3b

We recently demonstrated that herpes simplex virus type 1 (HSV 1) induces a receptor on human umbilical vein endothelial cells for complement component C3b (C3bR). We assigned this receptor function to HSV 1 viral glycoprotein C (gC) based on several observations: tunicamycin, which prevents glycosylation and expression of N-linked glycoproteins on the surface of infected cells, markedly reduced expression of the C3bR; monoclonal antibodies to HSV 1 gC blocked detection of the C3bR, whereas monoclonal antibodies to other HSV 1 glycoproteins (gB, gD, gE) had no effect; and the MP mutant of HSV 1, which fails to express gC, did not induce C3bR. We now report that HSV 1 induces C3bR on a wide variety of cell types including bovine thoracic aorta and pulmonary artery endothelial cells, human embryonic lung and embryonic foreskin fibroblasts, and human embryonic kidney cells. To date, all cells studied that are permissive to HSV 1 express C3bR, although the pattern of rosetting of C3b-coated erythrocytes varies among the cell strains examined. We also demonstrate that C3bR expression is not a general response of human umbilical vein endothelial cells to injury, because three other viruses (adenovirus 7, measles, and mumps) do not induce C3bR after infection of these cells. Previously we had shown that among herpes simplex viruses, a variety of HSV 1 strains induce C3bR, whereas HSV 2 strains do not. We now demonstrate that other herpes family viruses (CMV and VZV) do not express C3bR. Therefore, C3bR expression appears to be unique for HSV 1 and occurs on a wide variety of cells permissive to this virus.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.