Structure of a protein photocycle intermediate by millisecond time-resolved crystallography

The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle. The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy. Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds. An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen. Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction. The structural results suggest a general framework for the interpretation of protein photocycles.     

Structure of a sarcoplasmic calcium-binding protein from amphioxus refined at 2.4 A resolution

The three-dimensional structure of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus has been determined at 2.4 A resolution using multiple-isomorphous-replacement techniques. The refined model includes all 185 residues, three calcium ions, and one water molecule. The final crystallographic R-factor is 0.199. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.015 A and 2.8 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core, unlike the extended dumbbell-shaped structures of calmodulin or troponin C. There are four distinct domains with the typical helix-loop-helix Ca(2+)-binding motif (EF hand). The conformation of the pair of EF hands in the N-terminal half of the protein is unusual due to the presence of an aspartate residue in the twelfth position of the first Ca(2+)-binding loop, rather than the usual glutamate. The C-terminal half of the molecule contains one Ca(2+)-binding domain with a novel helix-loop-helix conformation and one Ca(2+)-binding domain that is no longer functional because of amino acid changes. The overall structure is quite similar to a sarcoplasmic Ca(2+)-binding protein from sandworm, although there is only about 12% amino acid sequence identity between them. The similarity of the structures of these two proteins suggests that all sarcoplasmic Ca(2+)-binding proteins will have the same general conformation, even though there is very little conservation of primary structure among the proteins from various species.     

pH dependence of the formation of an M-type intermediate in the photocycle of 13-cis-bacteriorhodopsin

Phosphatidic acid (PA) dose-dependently induced superoxide (O2-) production of electropermeabilized human neutrophils but not of intact neutrophils, indicating that PA induces the activation of NADPH oxidase by acting on an intracellular target. The O2- production by PA was not inhibited by protein kinase C (PKC) inhibitors, such as staurosporine and calphostin C, and an inhibitor of PA phosphohydrolase, propranolol. These observations suggest that the activation of the oxidase by PA is independent of the activity of PKC and may dominate the activation by diacylglycerol which is formed from PA via the action of PA phosphohydrolase. Furthermore, the production by PA, as well as that by phorbol myristate acetate, was inhibited by cyclic AMP and GDP beta S. Therefore, PA seems to act at a site downstream of PKC.     

Interaction between Asp-85 and the proton-releasing group in bacteriorhodopsin. A study of an O-like photocycle intermediate

Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted O1. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these O1 species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid-base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The O1 species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in O1, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of O1. At high pH (>9), O1 reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (> 11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pKa values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pKa values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the O1 species, due to the inhibition of thermal trans-13-cis isomerization. The results are relevant to the molecular mechanism of the photocycles of both 13-cis- and all-trans-bR, primarily to the nature and to the deprotonation mechanism of the proton-releasing group.     

Structure of Sesbania mosaic virus at 4.7 A resolution and partial sequence of the coat protein

Serum IgG subclasses and Serum IgA were studied in 43 infants with acute bronchiolitis and 20 healthy infants. IgG subclasses were determined by a capture ELISA and IgA was quantified by turbidimetry. IgG1 concentrations were significantly lower in infants with bronchiolitis than in normal infants. The other IgG subclasses and IgA did not differ between the groups. The subgroups of infants with bronchiolitis who had previously suffered from otitis media or bronchitis, had significantly lower IgG2 than the other infants with bronchiolitis. The same was found for infants with bronchiolitis who had suffered from three or more lower respiratory tract infections. In infants who had suffered from upper or lower respiratory infections before the acute bronchiolitis, IgA was significantly higher than in infants without previous respiratory infections. Ten infants with bronchiolitis (23%) had IgG1 deficiency, that is values below the lower reference limit calculated in a population of healthy Norwegian infants. No healthy infants had any IgG1 deficiency. No infant with bronchiolitis had IgG2 or IgG3 deficiency. The low IgG1 values found in infants with acute bronchiolitis, may be one cause for infants to be more susceptible to RS virus infections.     

Structure of an Fab fragment against a C-terminal peptide of hCG at 2.0 A resolution

3A2 is an antibody raised against human chorionic gonadotropin and recognizes a linear epitope on the C-terminal peptide of the human chorionic gonadotropin beta-subunit. Its three-dimensional structure has been determined to 2-A resolution using molecular replacement and refined to a conventional R-factor of 18.2%. The protein exhibits the typical immunoglobulin fold, and the model contains 944 ordered water molecules and one sulfate ion. A comparison of the complementarity-determining regions of the Fab3A2 with those from the Protein Data Bank following the canonical structure method reveals a canonical main chain conformation. This antibody belongs to the canonical structure class (combination of canonical conformations of the complementarity determining loops) that shows a preference for haptens and not for peptides. However, the shape of the surface of the antigen binding loops resembles that of an anti-peptide antibody.     

Characteristics of hydrolytic protein breakdown at an early age

Proteolysis of milk proteins occurring within the gastrointestinal tract was distinctly manifested in young rats during the period of milk feeding as shown by gel filtration of nitrogenous components from contents of various parts of small intestine of 3, 20 and 30 days old rats as well as by means of ion exchange chromatography of free amino acids and estimation of their concentration and the rate of the amino acids liberation after incubation of the content of these parts of the intestine in vitro. In 3-20 days old rats the cavitary proteolysis was competed in the distal part of small intestine, in adult animals--in the proximal part. The amino acids might be divided into two groups: the rapidly liberated substances (leucine, isoleucine, lysine, arginine, tyrosine, serine) and the slowly liberated compounds (proline, glutamic and aspartic acids). The data obtained suggest the existance of two types of protein digestion--cavitary and intracellular (and/or membrane type) during this period of the ontogenesis; concepts of alimentary specificity of caseins are also discussed.     

A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy

The rate-limiting step during the refolding of S54G/P55N ribonuclease T1 is determined by the slow trans-->cis prolyl isomerisation of Pro39. We investigated the refolding of this variant by one-dimensional (1D) and two-dimensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10 degreesC. Two intermediates could be resolved with the 1D approach. The minor intermediate, which is only present early during refolding, is largely unfolded. The major intermediate, with an incorrect trans Pro39 peptide bond, is highly structured with 33 amide protons showing native chemical shifts and native NOE patterns. They could be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spectroscopy) by using a new assignment strategy to generate positive and negative signal intensities for native and non-native NOE cross-peaks, respectively. Surprisingly, amide protons with non-native environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, including the C-terminal part of the alpha-helix, beta-strands 3 and 4 and several loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal beta-strands 1 and 2 and the C terminus (connected by the disulfide bonds), the N-terminal part of the alpha-helix, and the loops between beta-strands 4/5 and 5/6. Implications of these native and non-native structure elements of the intermediate for the refolding of S54G/P55N ribonuclease T1 and for cis/trans isomerizations are discussed.     

Structure of Peptococcus aerogenes ferredoxin. Refinement at 2 A resolution

The model for ferredoxin from Peptococcus aerogenes, derived from a 2.8-A resolution map, has been refined with a 2-A resolution data set. The conventional index, R, decreased from 0.449 for the initial model to 0.188 for the unconstrained one and 0.206 for the constrained one. The standard deviations of the iron and sulfur atoms are 0.04 to 0.05 A and for the oxygen, nitrogen, and carbon atoms they range from 0.14 to 0.26 A. One hundred forty-six water oxygen atoms were included in the solvent part of the model and were checked by a highly selective criterion, suggesting that most of them represented water molecules.     

A case of anorexia at an early age

We report a case of early-age anorexia for which no organic etiology has been found so far. This case raises many unsolved questions. Early-age anorexia is rarely described in medical literature. No nosographical consensus has been established. In front of these children who refused eating, it is important to confronted with consider the psychological and somatical aspects to prevent later effects on the cognitive development.     

Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 A resolution

BACKGROUND: Translation initiation factor 5A (IF-5A) is reported to be involved in the first step of peptide bond formation in translation, to be involved in cell-cycle regulation and to be a cofactor for the Rev and Rex transactivator proteins of human immunodeficiency virus-1 and T-cell leukemia virus I, respectively. IF-5A contains an unusual amino acid, hypusine (N-epsilon-(4-aminobutyl-2-hydroxy)lysine), that is required for its function. The first step in the post-translational modification of lysine to hypusine is catalyzed by the enzyme deoxyhypusine synthase, the structure of which has been published recently. RESULTS: IF-5A from the archebacterium Pyrobaculum aerophilum has been heterologously expressed in Escherichia coli with selenomethionine substitution. The crystal structure of IF-5A has been determined by multiwavelength anomalous diffraction and refined to 1.75 A. Unmodified P. aerophilum IF-5A is found to be a beta structure with two domains and three separate hydrophobic cores. CONCLUSIONS: The lysine (Lys42) that is post-translationally modified by deoxyhypusine synthase is found at one end of the IF-5A molecule in an turn between beta strands beta4 and beta5; this lysine residue is freely solvent accessible. The C-terminal domain is found to be homologous to the cold-shock protein CspA of E. coli, which has a well characterized RNA-binding fold, suggesting that IF-5A is involved in RNA binding.     

The structure of the membrane protein squalene-hopene cyclase at 2.0 A resolution

Squalene cyclases catalyze a cationic cyclization cascade, which is homologous to a key step in cholesterol biosynthesis. The structure of the enzyme from Alicyclobacillus acidocaldarius has been determined in a new crystal form at 2.0 A resolution (1 A=0.1 nm) and refined to an R-factor of 15.3 % (Rfree=18.7 %). The structure indicates how the initial protonation and the final deprotonation of squalene occur and how the transient carbocations are stabilized. The pathways of the flexible educt squalene from the membrane interior to the active center cavity and of the rigid fused-ring product hopene in the reverse direction are discussed. The enzyme contains eight so-called QW-sequence repeats that fortify the alpha/alpha-barrels by an intricate interaction network. They are unique to the known triterpene cyclases and are presumed to shield these enzymes against the released enthalpy of the highly exergonic catalyzed reaction. The enzyme is a monotopic membrane protein, the membrane-binding interactions of which are described and compared with those of two prostaglandin-H2 synthase isoenzymes, the only other structurally characterized proteins of this type. In the crystals the membrane-binding regions face each other, suggesting a micelle-type detergent structure between them.     

Structure and molecular model refinement of pig pancreatic alpha-amylase at 2.1 A resolution

The previously reported structural model of porcine pancreatic alpha-amylase has been corrected and improved by a genuinely independent structure solution. The electron density map was established by multiple isomorphous replacement (m.i.r.; using 5 derivatives) and subsequent solvent-flattening at 2.8 A resolution. The sequence was built into the well-defined regions of the m.i.r. map; this partial model was refined using a simulated annealing refinement method with phase restraints. Phase combination of m.i.r. phases and phases of the partial model allowed the completion of the model. The final refinement was based on 29,838 independent reflections in the 8 to 2.1 A resolution range. A final R-factor of 15.6% was obtained with a model obeying standard geometry within 0.014 A in bond lengths and 2.8 degrees in bond angles. The final model consists of all 496 amino acid residues, 1 calcium ion, 1 chloride ion and 353 water molecules. The model is described in detail; the calcium and chloride binding sites are characterized.     

Structure of a pancreatic alpha-amylase bound to a substrate analogue at 2.03 A resolution

The structure of pig pancreatic alpha-amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) soaked with an enzyme-resistant substrate analogue, methyl 4,4'-dithio-alpha-maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the "second binding site." The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite -1 (using a nomenclature where the site of hydrolysis is taken to be between subsites -1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the "flexible loop" that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 A resolution, to an R-factor of 16.0 (Rfree, 18.5).     

Structure of human des(1-45) factor Xa at 2.2 A resolution

The structure of a large molecular fragment of factor Xa that lacks only a Gla (gamma-carboxyglutamic acid) domain (N-terminal 45 residues) has been solved by X-ray crystallography and refined at 2.2 A resolution to a crystallographic R-value of 0.168. The fragment identity was clearly established by automated Edman degradation. X-ray structure analysis confirmed the biochemical characterization and also revealed that the N-terminal epidermal growth factor (EGF)-like domain is flexibly disordered in crystals. The second EGF module, however, is positionally ordered making contacts with the catalytic domain. The overall folding of the catalytic domain is similar to that of alpha-thrombin, excluding the insertion loops of the latter with respect to simpler serine proteinases. The C-terminal arginine of the A-chain interacts in a substrate-like manner with the S1 specificity site of the active site of a crystallographically neighboring molecule. Based on this interaction and the structure of D-PheProArg methylene-thrombin, a model of the commonly used dansylGluGlyArg methylene inhibitor-factor Xa interaction is proposed. The region of factor Xa corresponding to the fibrinogen recognition site of thrombin has a reversed electrical polarity to the anion binding fibrinogen recognition site of thrombin but possesses a site similar to the Ca2+ binding site of trypsin and other serine proteinases. The structure of the C-terminal EGF domain of factor Xa is the first to be determined crystallographically. Its folding has been comprehensively compared with similar domains determined by NMR. Although the A-chain makes 44 contacts at less than 3.5 A with the catalytic domain, only 16 involve the EGF module. In addition, the A-chain makes 30 intermolecular contacts with a neighboring catalytic domain.     

New insights into the photocycle of Ectothiorhodospira halophila photoactive yellow protein: photorecovery of the long-lived photobleached intermediate in the Met100Ala mutant

There are previously two known intermediates (I1 and I2) in the room-temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. The three-dimensional structures of ground-state PYP and of I2 have shown that light-induced conformational changes are localized to the active site. Previous site-specific mutagenesis studies of PYP in our laboratories have characterized two active site mutants (Glu46Gln and Arg52Ala). We now report the construction and characterization of a mutant at a third active site position (Met100Ala) in order to establish the role of this residue in the photocycle. Met100Ala PYP has an absorption spectrum which is very similar to wild-type (WT) PYP, but exhibits very different kinetic properties. At pH 7.0, the light-induced bleaching reaction (I2 formation) has a half-life <1 microseconds and the recovery in the dark has a half-life of 5.5 min, as compared with half-lives of 100 microseconds and 140 ms for the same reactions in WT PYP. The slow rate of recovery from I2 for Met100Ala results in the accumulation of the bleached intermediate even under room light illumination. These results are qualitatively similar to what has been observed with the Arg52Ala mutant of PYP, and with WT PYP in the presence of alcohols or urea, and suggest that Met100 acts to stabilize the ground state of the protein. The midpoint for guanidine denaturation confirms this. The slow recovery of I2 in the Met100Ala mutant has allowed us to obtain direct evidence that this intermediate species is also photoactive and can be returned to the ground state by a 365 nm laser flash, with kinetics (half-life = 160 microseconds; k = 6300 s-1) which are 6 orders of magnitude faster than dark recovery. This implies that chromophore reisomerization limits the rate of conversion of I2 to the ground state in PYP. Met100 is in van der Waals contact with the chromophore in the I2 state, and we suggest that the sulfur atom catalyzes cis-trans isomerization in WT PYP.     

Structure of barley grain peroxidase refined at 1.9-A resolution. A plant peroxidase reversibly inactivated at neutral pH

The crystal structure of the major peroxidase of barley grain (BP 1) has been solved by molecular replacement and phase combination and refined to an R-factor of 19.2% for all data between 38 and 1.9 A. The refined model includes amino acid residues 1-309, one calcium ion, one sodium ion, iron-protoporphyrin IX, and 146 solvent molecules. BP 1 has the apparently unique property of being unable to catalyze the reaction with the primary substrate hydrogen peroxide to form compound I at pH values > 5, a feature investigated by obtaining crystal structure data at pH 5.5, 7.5, and 8.5. Structural comparison shows that the overall fold of inactive barley grain peroxidase at these pH values resembles that of both horseradish peroxidase C and peanut peroxidase. The key differences between the structures of active horseradish peroxidase C and inactive BP 1 include the orientation of the catalytic distal histidine, disruption of a hydrogen bond between this histidine and a conserved asparagine, and apparent substitution of calcium at the distal cation binding site with sodium at pH 7.5. These profound changes are a result of a dramatic structural rearrangement to the loop region between helices B and C. This is the first time that structural rearrangements linked to active site chemistry have been observed by crystallography in the peroxidase domain distal to heme.     

Enantioselective synthesis of a key intermediate of 20(S)-camptothecin via an enzyme-catalyzed resolution

The key intermediate of a 20(S)-camptothecin 1 synthesis was obtained in a highly enantioselective fashion using an enzyme-catalyzed resolution. A commercially available protease was found to exhibit the highest enantioselectivity with moderate activity, and (S)-ethyl 2-acetoxy-2-[6-(acetoxymethyl)-1,1-(ethylenedioxy)-5-oxo- 1,2,3,5-tetrahydroindolizin-7-yl]butanoate 7c of 98% e.e. was obtained as the remaining substrate.