酶联免疫分析法探测Cry1Ab蛋白在不同介质中的构象变化

采用酶联免疫分析（ELISA）方法,根据构象变化后的蛋白与抗体结合能力下降从而导致ELISA测定值降低的原理,探测了转cry1Ab基因水稻表达的Cry1Ab蛋白在不同溶液介质中的热致构象变化行为,以及不同有机溶剂及溶液pH值对该蛋白构象变化的影响程度。实验表明,Cry1Ab蛋白在不同条件下的构象变化程度可以灵敏地通过ELISA方法检测。在不同的介质中,CrylAb蛋白的热致构象变化程度不同。在Na2SO4介质中,该蛋白具有较高的热稳定性;SDS的存在,可以促进该蛋白的构象变化。常温下,25％（V／V）的有机溶剂乙腈、异丙醇、甲醇、乙醇均能使该蛋白的构象发生转变,其中以乙腈最为显著。醇类溶剂对Cry1Ab蛋白的构象影响程度随疏水性增大而增大;溶液pH值也对该蛋白的构象变化产生影响。pH在8-10之间,该蛋白构象能保持稳定;酸或过碱性的溶液均能使蛋白构象偏离原始状态,从而引起ELISA测定值的降低。另外,腐殖酸能在一定时间内保持Cry1Ab蛋白构象的稳定性。     

Fine-tuning the pH trigger of self-assembly

The creation of smart, self-assembling materials that undergo morphological transitions in response to specific physiological environments can allow for the enhanced accumulation of imaging or drug delivery agents based on differences in diffusion kinetics. Here, we have developed a series of self-assembling peptide amphiphile molecules that transform either isolated from molecules or spherical micelles into nanofibers when the pH is slightly reduced from 7.4 to 6.6, in isotonic salt solutions that simulate the acidic extracellular microenvironment of malignant tumor tissue. This transition is rapid and reversible, indicating the system is in thermodynamic equilibrium. The self-assembly phase diagrams show a single-molecule-to-nanofiber transition with a highly concentration-dependent transition pH. However, addition of a sterically bulky Gd(DO3A) imaging tag on the exterior periphery shifts this self-assembly to more acidic pH values and also induces a spherical micellar morphology at high pH and concentration ranges. By balancing the attractive hydrophobic and hydrogen-bonding forces, and the repulsive electrostatic and steric forces, the self-assembly morphology and the pH of transition can be systematically shifted by tenths a pH unit.     

Isothermal titration calorimetric studies of the pH induced conformational changes of bovine serum albumin

Bovine serum albumin (BSA) is a soft globular protein that undergoes conformational changes through several identified transition steps in the pH range 2–13.5. The ability to change conformation makes BSA capable of complexing different ligands from fatty acids to cations or drugs and carries them in the bloodstream. Microcalorimetric titration of BSA with NaOH solution was performed to measure the enthalpy of conformational changes. Two exothermic enthalpy changes were found in the course of the titration between pH 3 and 9.5, which can be identified with the E–F, and the F–N transitions. The enthalpy change at pH 3.5 (transition from the E to the F form of BSA, folding of intra-domain helices in domain I) is independent of the protein concentration. The second transition (F–N, folding of domain III) was observed at pH 4.8 for the 0.1% BSA solution, but it shifted to higher pH values as the protein concentration increased to 0.2% and 0.3%. The tightening of the protein structure with increasing pH was verified measuring intrinsic fluorescence of tryptophan residues. At even higher pH value, pH 10.5, fluorescence measurements revealed protein expansion. The BSA conformational changes were also measured by dynamic light scattering. The hydrodynamic diameter was smaller at the i.e.p. of BSA (5–7 nm at pH ~5) and larger at the two ends of the pH range (17.5 nm at pH 2 and 8.3 nm at pH 10).     

TIME-RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR-185→E: ASP-96 REPROTONATES DURING O FORMATION; ASP-85 AND ASP-212 DEPROTONATE DURING O DECAY

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.     

Alternative mechanistic explanation for ligand-dependent selectivities in copper-catalyzed N- and O-arylation reactions

The ligand-dependent selectivities in Ullmann-type reactions of amino alcohols with iodobenzene by β-diketone- and 1,10-phenanthroline-ligated Cu(I) complexes were recently explained by the single-electron transfer and iodine atom transfer mechanisms (Jones, G. O., Liu, P., Houk, K. N., and Buchwald, S. L. J. Am. Chem. Soc. 2010, 132, 6205.). The present study shows that an alternative, oxidative addition/reductive elimination mechanism may also explain the selectivities. Calculations indicate that a Cu(I) complex with a negatively charged β-diketone ligand is electronically neutral, so that oxidative addition of ArI to a β-diketone-ligated Cu(I) prefers to occur (and occur readily) in the absence of the amino alcohol. Thus, coordination of the amino alcohol in its neutral form can only occur at the Cu(III) stage where N-coordination is favored over O-coordination. The coordination step is the rate-limiting step and the outcome is that N-arylation is favored with the β-diketone ligand. On the other hand, a Cu(I) complex with a neutral 1,10-phenanthroline ligand is positively charged, so that oxidative addition of ArI to a 1,10-phenanthroline-ligated Cu(I) has to get assistance from a deprotonated amino alcohol substrate. This causes oxidative addition to become the rate-limiting step in the 1,10-phenanthroline-mediated reaction. The immediate product of the oxidative addition step is found to undergo facile reductive elimination to provide the arylation product. Because O-coordination of a deprotonated amino alcohol is favored over N-coordination in the oxidative addition transition state, O-arylation is favored with the 1,10-phenanthroline ligand.     

SPECTROSCOPIC INVESTIGATION OF DIHYDRONICOTINAMIDES-I: CONFORMATION, ABSORPTION, AND FLUORESCENCE

Abstract— The 1(N)-(2,6-dichlorobenzyl)-1,4-dihydronicotinamide (I), N-methyl- and N,N-dimethyl-1(N)-(2,6-dichlorobenzyl)-1,4-dihydronicotinamide (II and III), respectively), and 1(N)-(2,6-dichloro-benzyl)-2-aminomethyl-1,4-dihydronicotinic acid lactame (IV) were synthesized as model compounds for natural coenzymes, and systematically studied by 1H NMR, UV/V1S absorption and fluorescence spectroscopy. The absorption at ∼ 340 nm argues for an effective conjugation between dihydropyridine and carboxamide π-system, and rules out any severely twisted conformation. For the natural coenzymes NADH and NMNH, as well as for I and II (with no or only one N-amide substituent), 1H NMR definitively establishes a transoid conformation in solution, with the carbonyl O close to 2-H of the dihydropyridine ring. N,N-dimethyl substitution effectively inverts the carboxamide orientation into the cisoid form. The 1H NMR data (as well as molar extinctions) for the fused-ring derivatives IV and V, with a fixed cisoid and transoid structure, respectively, provide final proof for the conformational assignment.
Absorption maxima are shifted to lower energies with increasing solvent polarity. In solvents which can act as hydrogen bond acceptors to the carboxamide N-H, absorption shows a general blue-shift of ∼ 10 nm. H-bond donor solvents do not affect absorption maxima but enhance molar extinction. Fluorescence maxima show a similar dependence on solvent polarity but no specific hydrogen-bonding effect. Fluorescence quantum yields appear increased tenfold in solvents donating H-bonds to the carboxamide C=O group. These results are interpreted in terms of the vinylogous amide resonance between C=O function and ring-N lone pair being the electronic interaction dominating in the ground state of dihydronicotinamides.     

Stable conformations of tripeptides in aqueous solution studied by UV circular dichroism spectroscopy

Determination of the precise solution structure of peptides is of utmost importance to the understanding of protein folding and peptide drugs. Herein, we have measured the UV circular dichroism (UVCD) spectra of tri-alanine dissolved in D(2)O, H(2)O, and glycerol. The results clearly show the coexistence of a polyproline II or 3(1)-helix and a somewhat disordered flat beta-strand conformation, in complete agreement with recent predictions from spectroscopic data (Eker et al. J. Am. Chem. Soc. 2002, 124, 14 330-14 341). A thermodynamic analysis revealed that enthalpic contributions of about 11 and 17 kJ/mol stabilize polyproline II in D(2)O and H(2)O, respectively, but at room temperature they are counterbalanced by entropic contributions, which clearly favor the more disordered beta-strand conformation. It is hypothesized that this delicate balance is the reason for the variety of structural propensities of amino acid residues in the absence of nonlocal interactions. The isotope effect yielding a higher occupation of polyproline II in H(2)O with respect to D(2)O strongly suggests that a hydrogen-bonding network involving the peptide and water molecules in the hydration shell plays a major role in stabilizing this conformation. The equilibrium between polyproline II and beta-strand is practically maintained in glycerol, which suggests that glycerol can substitute water as stabilizing solvent for the polyproline II conformation. We also measured the UVCD spectra of tri-valine and tri-lysine (both at acidic pD) in D(2)O and found them to adopt a flat beta-strand and left-handed turn structure, respectively, in accordance with recent analyses of vibrational spectroscopy data. Generally, the present study adds substantial evidence to the notion that the so-called random coil state of peptides is much more structured than generally assumed.     

Acid-Catalyzed Amine-Borane Reduction of Nitrite

The rate of reduction of nitrite by trimethylamine-borane was followed by observing the decrease in nitrite absorbance under pseudo-first-order conditions. The reaction is acid-catalyzed and exhibits a first-order dependence on both amine-borane and total nitrite concentration. The molar equivalence of NaNO(2) to (CH(3))(3)NBH(3) = 2:1. Equimolar amounts of hydrogen and nitrous oxide are formed, and the molar ratio of nitrite reacted to N(2)O produced is 2:1. In concentrated HCl or H(2)SO(4), a correlation of rate with the Hammett acidity function, h(o), is observed. The reaction is subject to a pronounced inversesolvent isotope effect (k(D)()2(O)/k(H)()2(O) approximately 2.7) and a modest normal substrate effect (k((CH)()3())()3(N.BH)()3/k((CH)()3())()3(N.BD)()3 approximately 1.4). The reaction is first-order in H(3)O(+) in the region pH 0.7-2.7, but a second-order dependence is observed above pH 4 with the transition occurring at pH approximately pK(a) for HNO(2). Results are consistent with a mechanistic model involving preequilibration protonation of molecular nitrous acid followed by rate-limiting hydride attack on H(2)ONO(+) or free NO(+) to produce nitrosyl hydride as a reactive intermediate.     

Polymorphic transitions in n-hydrocarbon-water and n-alcohol-water binary systems

The mixing of hydrocarbons and alcohols in an excess of water is explained by polymorphic transitions similar to crystallization in an ensemble of water clusters. Enthalpies of transitions of 4.90 ± 0.07 and 2.2 ± 0.3 kJ/mol are obtained for solutions of hydrocarbons and alcohols in an excess of water, respectively. It is concluded that the mixing of water in an excess of hydrocarbons and alcohols is similar to evaporation (the breaking of H-bonds) with an enthalpy of 34 ± 1.4 kJ/mol. It is established that a polymorphic transition occurs between two binodals, and is accompanied by the emergence of microphases (concentration fluctuations) of alcohols in water. Binodals and spinodals in an excess of water and alcohol coincide for butyl and other higher alcohols.     

pH值对丝素蛋白构象转变的影响

模仿家蚕吐丝过程中伴随丝素蛋白自然脱水的纤维化过程,研究了再生丝素蛋白在各种pH值的磷酸盐缓冲溶液体系中自然干燥脱水成膜后的构象转变.利用激光拉曼散射光谱及其二维相关光谱,定性分析了丝素蛋白酰胺区(1600～1700cm^-1)散射峰的相关组成及结构.在此基础上,利用¹³CCP-MAS固体核磁共振谱对丝素蛋白丙氨酸C_β峰(δ14.5～22)进行了解析拟合.从而确定了体系中与Silk及Silk构象相关的组成含量与pH值的关系.结果表明,pH=5.2的酸性溶液有利于蚕丝丝素蛋白从Silk向Silk构象转变,而中性与碱性溶液(pH=6.9和8.0)则对丝素蛋白的构象转变影响甚小.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP

Abstract
Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The p K values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

Zeta potential of microbubbles in aqueous solutions: electrical properties of the gas-water interface

Microbubbles are very fine bubbles and appropriate for the investigation of the gas-water interface electrical charge, because of their long stagnation, due to slow buoyancy, in the electrophoresis cell observation area. This study investigated the zeta potential of microbubbles in aqueous solutions and revealed that the bubbles were negatively charged under a wide range of pH conditions. The potential was positive under strong acidic conditions, and the inorganic electrolytes decrease the potential by increasing the amount of counterions within the slipping plane. OH(-) and H(+) are crucial factors for the charging mechanism of the gas-water interface, while other anions and cations have secondary effects on the zeta potential, because counterions are attracted by the interface charge. The addition of a small amount of propanol and butanol provided significant information for considering the mechanism of the gas-water interface charge. Even though these alcohols did not have any electrical charge, they had a strong effect on the gas-water interface charge and dispersed the zeta potential of the microbubbles in the aqueous solution. These alcohols tended to adsorb to the interface and affect the hydrogen-bonding network at the interface, so that it was concluded that the gas-water interface electrical charge must be related to the difference of the construction of the hydrogen-bonding network between the bulk water and the gas-water interface.     

过渡金属甲基磺酸盐催化醇的四氢吡喃化反应

以过渡金属甲基磺酸盐［Mn(CH₃SO₃)₂·2H₂O, Cu(CH₃SO₃)₂·4H₂O, Co(CH₃SO₃)₂·4H₂O和Zn(CH₃SO₃)₂·4H₂O］为催化剂,在室温条件下催化醇的四氢吡喃化反应,并对反应条件进行了优化。结果表明：当醇用量为30 mmol,醇和3,4-二氢吡喃摩尔比为1.0 ：1.1,甲基磺酸盐用量为1 mmol,二氯甲烷20 mL时,可高效催化醇的四氢吡喃化反应。与路易斯酸催化活性相比,过渡金属甲基磺酸盐催化醇的四氢吡喃化反应效果最好,催化酚的效果较差。用Mn(CH₃SO₃)₂·2H₂O和Cu(CH₃SO₃)₂·4H₂O催化正丁醇的四氢吡喃化反应,重复使用5次,收率分别为89%和92%。     

Mass spectrometry and hydrogen/deuterium exchange measurements of alcohol-induced structural changes in cellular retinol-binding protein type I

To bind and release its ligand, cellular retinol-binding protein type I (CRBP) needs to undergo conformational and dynamic changes to connect the inner, solvent-shielded cavity, where retinol is found to bind, and the outside medium. Retinol dissociation in vitro is favoured by water/alcohol mixtures whose moderately low dielectric constants mimic a property characteristic of the membrane microenvironment where this process occurs in vivo. Apo- and holo-CRBP, in either water/methanol or water/trifluoroethanol (TFE) mixtures, were analyzed at equilibrium by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) to identify the alcohol-induced species. The questions were asked whether the presence of alcohols affects protein dynamics, as reflected by hydrogen/deuterium (H/D) exchange monitored by continuous-labelling experiments, and to which extent retinol dissociation influences the process. With increasing methanol, at pH near neutrality, apo-CRBP exhibits a progressively more compact conformation, resulting in reduced H/D exchange with respect to the native protein in water. Retinol dissociation from the holo-protein did not promote hydrogen replacement. Similarly, in the presence of the low TFE concentration sufficient to cause retinol dissociation, the hydrogen exchange of the resulting apo-protein was not exalted. However, in contrast with the alkanol, higher TFE concentrations induced a transition of apo-CRBP to a new alpha-helix conformation capable of exchanging all available hydrogen atoms.     

Physical and kinetic analysis of the cooperative role of metal ions in catalysis of phosphodiester cleavage by a dinuclear Zn(II) complex

A dinuclear metal ion complex Zn(2)()(L2O) and its mononuclear analogue Zn(L1OH) were synthesized and studied as catalysts of the cleavage of the phosphate diester 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). X-ray crystal structure data, potentiometric titrations, and (1)H NMR spectra obtained over a wide range of pH values provide strong evidence that the alcohol linker in the complex Zn(2)()(L2O) is ionized below pH 6.0, while the alcohol group in the complex Zn(L1OH) remains protonated even at high pH. The ionizations observed at high pH correspond to the formation of the monohydroxo complexes, Zn(2)(L2O)(OH) and Zn(L1OH)(OH), with pK(a)'s of 8.0 and 9.2, respectively. The pH-rate profiles of second-order rate constants for metal-ion complex-catalyzed cleavage of HPNP are reported. These show downward curvature centered at the pK(a)'s for the respective zinc-bound waters, and limiting second-order rate constants at high pH of k(c) = 0.71 M(-)(1) s(-)(1) for Zn(2)()(L2O) and 0.061 M(-)(1) s(-)(1) for Zn(L1OH). The larger catalytic activity of Zn(2)()(L2O) compared with Zn(L1OH) is due to the cooperative role of the metal ions in facilitating the formation of the ionized zinc-bound water at close to neutral pH and in providing additional stabilization of the rate-limiting transition state for phosphodiester cleavage. Zn(2)()(L2O) complex (1 M) at pH 7.6 stabilizes the transition state for the uncatalyzed reaction by 9.3 kcal/mol. Assuming that the dissociation constant determined for a diethyl phosphate inhibitor is similar to that for substrate, then ca. 2.4 kcal/mol of these stabilizing interactions are expressed in the ground-state Michaelis complex, while the bulk of these interactions are only expressed as the reaction approaches the transition state for phosphodiester cleavage.     

Heat capacities of butanol and pentanol in aqueous dodecyltrimethylammonium bromide solutions

Heat capacities of the ternary systems water-dodecyltrimethylammonium bromide (DTAB)-butanol and water-DTAB-pentanol were measured at 25°C. The standard partial molar heat capacities of pentanol in micellar solutions show a maximum at about 0.35 mol-kg^–1 DTAB that has been attributed to a micellar structural transition. This maximum tends to vanish by increasing the alcohol concentration and by decreasing the alcohol alkyl chain length; in the case of butanol it was not detected. The behavior of the standard partial molar heat capacities of alcohols in micellar solutions in the region above the cmc and below the structural transition was explained using a previously reported mass-action model for the alcohol distribution between the aqueous and the micellar phase and the pseudophase transition model for micellization. In the resulting equation the contributions due to the temperature effect on the shift of both the micellization equilibrium and the distribution are shown to be negligible so that only the distribution effect and the shift of the micellization equilibrium due to the added alcohol remain. The distribution constant and the partial molar heat capacities of alcohols in the aqueous and micellar phases have been derived by linear regression. The distribution constant for both alcohols agree well with those previously obtained using different techniques. Since the best fit below the structural transition correlates as well with the experimental points above the structural transition, it seems that no difference exists in the standard partial molar heat capacities of alcohols in the two shapes of the micelles. Also, from the present data and those for alkanols in sodium dodecylsulfate reported in the literature it seems that the standard heat capacity of alcohols in the micellar phase does not depend on both the alcohol alkyl chain length and the nature of the hydrophilic moiety of the head group of the micelles.     

THE INWARD H+ PATHWAY IN BACTERIORHODOPSIN: THE ROLE OF M412 AND P(N)560 INTERMEDIATES*

Abstract— In purple bacteriorhodopsin sheets adsorbed onto the phospholipid-impregnated collodion film, electrogenic stages are identified correlating with decays of the M and N(P)-type intermediates. It is concluded that both M → N and N → bR transitions are electrogenic.
The M decay is shown to be of a complex kinetics. In purple sheets, the lower the light intensity, the higher the rate of "slow M" decay. Such a dependence, which is absent from monomeric bacteriorhodopsin in proteoliposomes and from Triton X-100-solubilized protein, may be explained by the inhibiting effect of a light-induced conformation change in a bacteriorhodopsin molecule upon the M decay in some other bacteriorhodopsin molecules within the same sheet.
The light intensity-independent "slow M" decay in solubilized bacteriorhodopsin is shown to correlate with the decay of the N intermediate and H⁺ uptake after the flash. In contrast to "fast M", "slow M" is pH dependent, closely resembling in this respect the N intermediate. It is suggested that there is a fast light-independent equilibration between M and N so that "slow M" represents the portion of the M pool that monitors the N concentration. The M → N equilibrium is assumed to be involved in the effect of the light-induced electric field on the M decay. No direct effect of light on the equilibrium was found.     

Novel chiral thiazoline‐containing N―O ligands in the asymmetric addition of diethylzinc to aldehydes: substituent effect on the enantioselectivity

Several novel chiral thiazoline primary and tertiary alcohols were easily synthesized from commercially available l ‐cysteine in three steps and with high yield. These ligands were subsequently applied to the asymmetric addition of diethylzinc (Et₂Zn) to various aldehydes. Products with S configuration were obtained when thiazoline‐containing tertiary alcohol ligands were used as catalysts. The primary alcohol induced corresponding products with R configuration in 68% enantiomeric excess, which was a higher value relative to other N―O ligands possessing a primary alcohol unit in the literature. Furthermore, a plausible transition state model was proposed to explain the observed enantioselectivities. Copyright © 2012 John Wiley & Sons, Ltd.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP*

Abstract— Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The pK values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

Rational modulation of the periodicity in linear hydrogen-bonded assemblies of trimesic acid on surfaces

We demonstrate a surprising cooperative adsorption process at the liquid-solid interface, involving self-assembly in which a three-fold hydrogen-bonding unit (trimesic acid, TMA) is forced into a linear pattern by noncovalent interaction with an alcohol. Our work shows that the unexpected linear pattern formed by coadsorption of TMA and alcohols can be modulated in size by choosing alcohols with different chain lengths.