bluuebunny/crossref_metadata_2025_split huggingface dataset:
Theory of chaperonin action: Inertial model for enhancement of prokaryotic rubisco assembly
Authors: Roy Harry , Kupferschmid Michael , Bell Jeffrey A. 
Published: February 2009

AbstractWe have performed a computational simulation of the aggregation and chaperonin‐dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P. Goloubinoff et al. (1989, Nature 342, 884–889) and P.V. Viitanen et al. (1990, Biochemistry 29, 5665–5671). The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding intermediate, Rubisco‐I, with itself and with aggregates of Rubisco‐I, leading up to dodecamers. Four rate constants, applying to forward or reverse steps in the aggregation process, were included. Optimal values for these constants were determined using the ellipsoid algorithm as implemented by one of us (Ecker, J.G. & Kupferschmid, M., 1988, Introduction to Operations Research, Wiley, New York, pp. 315–322). Intensive exploration of simpler aggregation models did not identify an alternative that could simulate the data as well as this one. The activity of the chaperonin in this system was simulated by using this aggregation model, combined with a model similar to that proposed by Goloubinoff et al. (1989). The model assumes that the chaperonin can bind the folding intermediate rapidly, and that the chaperonin complex releases the Rubisco molecule slowly, permitting time for its spontaneous folding while interacting with the chaperonin. This is followed by self‐association of the folded Rubisco monomer to yield the active dimeric Rubisco. A modification of the model that simulates temperature effects was also constructed. The most important results we obtained indicate that the chaperonin‐dependent reconstitution of Rubisco can be simulated adequately without invoking any catalysis of folding by the chaperonin. In addition, the simulations predict values for the association rate constant of Rubisco‐I with the chaperonin, and other variables, that are subject to experimental verification.
Analysis of peptide mixtures by capillary high performance liquid chromatography: A practical guide to small‐scale separations
Authors: Davis Michael T. , Lee Terry D. 
Published: February 2009

AbstractCapillary HPLC is a very effective means of separating small amounts of peptides and proteins. Capillary columns ranging from 0.01 mm to 0.5 mm in diameter can be constructed using recycled supports and inexpensive fused silica capillary tubing. Commercial pumping systems and UV detectors can be readily converted for operation in the flow rate range of 0.5–50 μL/min. Detailed procedures are given for the construction of columns and UV detector flow cells. A mixture of peptides derived from the endo Lys C digest of horse heart cytochrome c was used to illustrate various aspects of capillary chromatography of peptides and compare the performance of various‐sized capillary columns and UV detector flow cell types.
Overexpression, purification, and characterization of yeast cyclophilins A and B
Authors: Zydowsky Lynne D. , Ho Susanna I. , Baker C. Hunter , Walsh Christopher T. , Mcintyre Kim 
Published: February 2009

AbstractTwo isoforms of yeast cyclophilins, yCyPA and yCyPB, have been subcloned, expressed in Escherichia coli, and purified to homogeneity. The full‐length (163‐amino acid) yeast CyPA was easily expressed and purified; however, only a genetically truncated, 186‐residue form of yCyPB lacking a putative 20‐amino acid signal sequence could be purified. Each yeast cyclophilin isoform is a peptidyl‐prolyl isomerase, inhibitable by the immunosuppressive drug CsA (IC50's of 40 ± 8 nM and 101 ± 14 nM at 18 nM concentrations of yCyPA and yCyPB, respectively). Polyclonal antibodies raised against recombinant yCyPA detected native yCyPA in yeast cell extracts by both immunoprecipitation and Western blot analysis. However, polyclonal antibodies raised against recombinant yCyPB detected no native yCyPB in yeast cell extracts by Western blot analysis; small amounts of yCyPB were found in the culture broth, suggesting secretion extracellularly of this isoform. Northern analysis indicated that both yCyPA mRNA and yCyPB mRNA (at a much lower level) were detectable in cell‐free extracts. Characterization of the yeast cyclophilin proteins demonstrated that their catalytic properties and sensitivity to CsA parallel those of the human cyclophilins.
Intramolecular interactions in pancreatic ribonucleases
Authors: Kolbanovskaya Elena Y.U. , Karpeisky Marat Y.A. , Sathyanarayana Bangalore K. , Wlodawer Alexander 
Published: February 2009

AbstractA detailed analysis of the composition and properties of hydrophobic nuclei and microclusters in pancreatic ribonuclease A (RNase A) has been carried out. Distance calculations for all noncovalently bonded atoms revealed that the average number of nonpolar contacts between a side chain of an amino acid and its neighbors is substantially larger if it involves hydrophobic residues rather than nonhydrophobic ones. However, the difference decreased when the number of contacts per nonpolar group and/or atom were calculated. Three main nuclei and five microclusters were identified, and their quantitative parameters were calculated. These nuclei include hydrophobic residues with a substantial number of nonpolar contacts with the environment (Phe 8, Phe 120, Phe 46, Tyr 25, Tyr 97, Ile 107, Leu 35, Ile 81, Val 54, Val 108, Met 29, Met 30). Hydrophobic nuclei of RNase A differ in shape and in composition, in the number of intranuclear contacts and of associated residues, as well as in their internal mobility. All eight cysteine residues are involved in nonpolar interactions with amino acid residues of hydrophobic nuclei. Active site amino acid residues of RNase A form a noncovalent contact network comprised of themselves, as well as of many conserved residues from hydrophobic nuclei. Sequence alignment with some other members of the RNase A family of proteins shows remarkable similarity in positions and in conservation of the main nonpolar residues, comprising cores of two (out of three) hydrophobic nuclei. A correlation was shown to exist between the average density of contacts for side‐chain atoms and the number of amino acids to be found in the appropriate positions in the sequences of related mammalian ribonucleases. However, there are certain amino acid positions in the third, smaller nucleus, which are highly variable within the family. Taking into account that this nucleus is composed of residues belonging to different elements of the secondary structure, it is likely that the mutual orientation of these elements can be somehow different for these proteins.
Kinetic properties of tetrameric glycogen phosphorylase b in solution and in the crystalline state
Authors: Leonidas D. D. , Oikonomakos N. G. , Papageorgiou A. C. , Sotiroudis T. G. 
Published: February 2009

AbstractR‐state monoclinic P21 crystals of phosphorylase have been shown to be catalytically active in the presence of an oligosaccharide primer and glucose‐1‐phosphate in 0.9 M ammonium sulfate, 10 mM β‐glycerophosphate, 0.5 mM EDTA, and 1 mM dithiothreitol, the medium in which the crystals are grown or equilibrated for crystallographic studies (Barford, D. & Johnson, L.N., 1989, Nature 360, 609–616; Barford, D., Hu, S.‐H., & Johnson, L.N., 1991, J. Mol. Biol. 218, 233–260). Kinetic data suggest that the activity of crystalline tetrameric phosphorylase is similar to that determined in solution for the enzyme tetramer. However, large differences were found in the maximal velocities for both oligosaccharide or glucose‐1‐phosphate substrates between the soluble dimeric and crystalline tetrameric enzyme.
Structure of a rapidly formed intermediate in ribonuclease T1 folding
Authors: Kiefhaber Thomas , Schmid Franz Xaver , Willaert Kathrien , Engelborghs Yves , Chaffotte Alain 
Published: February 2009

AbstractKinetic intermediates in protein folding are short‐lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa‐Pro peptide bonds the final rate‐limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead‐time of stopped‐flow mixing (15 ms), indicating a fast formation of the single α‐helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead‐times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the β‐sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38‐proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (τ = 6,500 s at pH 5.0 and 10 °C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native‐like properties.
Correlation functions as a tool for protein modeling and structure analysis
Authors: BÖhm Gerald , Jaenicke Rainer 
Published: February 2009

AbstractProteins present unique folding structures whose conformations are determined primarily by their amino acid sequences. At present, there is no algorithm that would correlate the sequences with the structures determined by X‐ray analysis or NMR. Comparative modeling of a new protein sequence based on the known structure of a functionally related protein promises to yield model structures that may provide relevant properties of the protein. To analyze the quality of a model structure, a set of correlation functions was derived from calculations on a subset of proteins from the structure database. Twenty‐three highly resolved protein structures with resolutions of at least 1.7 Å from various protein families were used as the primary database. The purpose of this initial work was to find highly sensitive functions (including statistical error limits for the parameters) that describe properties of “real” proteins. Each correlation described is characterized by the correlation coefficient, the parameters for linear or nonlinear regression (coefficients of the equation), standard deviation and variance, and the confidence limits describing the statistical probability for values to occur within these limits, e.g., the natural variability of the property under examination. In addition, a method was developed for creating reasonably misfolded proteins. The ability of a correlation function to discriminate between the native structure and the misfolded conformations is expressed by the reliability index, which indicates the sensitivity of a correlation function. The term correlation functions thus summarizes a variety of efforts to find a mathematical description for the properties of protein structures, for their correlation, and for their significance.
Disulfide bridges in tomato pectinesterase: Variations from pectinesterases of other species; conservation of possible active site segments
Authors: Markoviĉ Oskar , Jörnvall Hans 
Published: February 2009

AbstractAnalysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys‐98 with Cys‐125, and Cys‐166 with Cys‐200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18–33% residue identities) from microorganisms. However, one half‐Cys (i.e., Cys‐166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N‐terminal part of the tomato pectinesterase, another in the C‐terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to highlight a functional site in a midchain segment.
Inactivation and covalent modification of CTP synthetase by thiourea dioxide
Authors: Robertson James G. , Sparvero Louis J. , Villafranca Joseph J. 
Published: February 2009

AbstractThiourea dioxide was used in chemical modification studies to identify functionally important amino acids in Escherichia coli CTP synthetase. Incubations at pH 8.0 in the absence of substrates led to rapid, time dependent, and irreversible inactivation of the enzyme. The second‐ order rate constant for inactivation was 0.18 M−1 s−1. Inactivation also occurred in the absence of oxygen and in the presence of catalase, thereby ruling out mixed‐function oxidation/reduction as the mode of amino acid modification. Saturating concentrations of the substrates ATP and UTP, and the allosteric activator GTP prevented inactivation by thiourea dioxide, whereas saturating concentrations of glutamine (a substrate) did not. The concentration dependence of nucleotide protection revealed cooperative behavior with respect to individual nucleotides and with respect to various combinations of nucleotides. Mixtures of nucleotides afforded greater protection against inactivation than single nucleotides alone, and a combination of the substrates ATP and UTP provided the most protection. The Hill coefficient for nucleotide protection was approximately 2 for ATP, UTP, and GTP. In the presence of 1:1 ratios of ATP:UTP, ATP:GTP, and UTP:GTP, the Hill coefficient was approximately 4 in each case. Fluorescence and circular dichroism measurements indicated that modification by thiourea dioxide causes detectable changes in the structure of the protein. Modification with [14C]thiourea dioxide demonstrated that complete inactivation correlates with incorporation of 3 mol of [14C]thiourea dioxide per mole of CTP synthetase monomer. The specificity of thiourea dioxide for lysine residues indicates that one or more lysines are most likely involved in CTP synthetase activity. The data further indicate that nucleotide binding prevents access to these functionally important residues.
Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K
Authors: Wong Wah Y. , Irvin Randall T. , Paranchych William , Hodges Robert S. 
Published: February 2009

AbstractThe C‐terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain‐specific monoclonal antibody PK99H, which blocks pilus‐mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134–140 (Asp‐Glu‐Gln‐Phe‐Ile‐Pro‐Lys) by using a single alanine replacement analysis on the 17‐residue synthetic peptide corresponding to the PAK C‐terminal sequence 128–144. Indeed, a 7‐residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17‐residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe‐Ile‐Pro‐Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain‐specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122–4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain‐specificity of PK99H for strains PAO, K122–4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain‐specificity of PK99H. Similarly, cross‐reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.
Total chemical synthesis, characterization, and immunological properties of an MHC class I model using the TASP concept for protein de novo design
Authors: Tuchscherer G. , Servis C. , Corradin G. , Blum U. , Rivier J. , Mutter M. 
Published: February 2009

AbstractThe design, total chemical synthesis, and immunological properties of a four‐α‐helix bundle template‐assembled synthetic protein (TASP) mimicking some of the structural features of the major histocompatibility complex (MHC) class I is described. In a first approach, the native sequence 58–74 of the α1 heavy chain domain of HLA‐A2 was modeled in order to increase helix stability and amphiphilicity of the 17‐mer peptide, preserving the residues for potential T‐cell receptor (TcR) binding properties. According to the TASP concept, these helical segments were covalently attached to a cyclic template molecule designed for the induction of a four‐helix‐bundle topology of the assembled peptide blocks.After extensive HPLC purification, stepwise solid‐phase synthesis resulted in a TASP molecule of high chemical purity as demonstrated by analytical HPLC, mass spectrometry, and amino acid analysis. CD spectroscopic investigations are consistent with the onset of a partial α‐helical conformation in aqueous buffer as well as in TFE. Antibodies raised directly against this four‐α‐helix bundle TASP molecule (without prior conjugation to a carrier molecule) were detected by ELISA. Flow cytometry studies showed that these antibodies recognize the native MHC class I molecule on the surface of HLA‐A2‐positive cells. The results indicate that the TASP approach represents a versatile tool for mimicking conformational epitopes.
Effects of DNA binding and metal substitution on the dynamics of the GAL4 DNA‐binding domain as studied by amide proton exchange
Authors: Mau Ted , Baleja James D. , Wagner Gerhard 
Published: February 2009

AbstractBackbone amide proton exchange rates in the DNA‐binding domain of GAL4 have been determined using 1H–15N heteronuclear correlation NMR spectroscopy. Three forms of the protein were studied — the native Zn‐containing protein, the Cd‐substituted protein, and a Zn‐GAL4/DNA complex. Exchange rates in the Zn‐containing protein are significantly slower than in the Cd‐substituted protein. This shows that Cd‐substituted GAL4 is destabilized relative to the native Zn‐containing protein. Upon DNA binding, global retardation of amide proton exchange with solvent was observed, indicating that internal fluctuations of the DNA‐recognition module are significantly reduced by the presence of DNA. In all forms of the protein, the internal dyad symmetry of the DNA‐recognition module of GAL4 is reflected by the backbone amide proton exchange rates.
Electronic and vibrational spectroscopy of the cytochrome c: Cytochrome c oxidase complexes from bovine and Paracoccus denitrificans
Authors: Lynch Stephen R. , Copeland Robert A. 
Published: February 2009

AbstractThe 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein‐chromophore systems, we suggest that the energy of the Soret π* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) π‐π interactions between the heme and aromatic amino acid residues.
A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41
Authors: Fujii Gary , Horvath Suzanna , Woodward Susan , Eiserling Fred , Eisenberg David 
Published: February 2009

AbstractThe mechanism of protein‐mediated membrane fusion and lysis has been investigated by solution‐state studies of the effects of peptides on liposomes. A peptide (SI) corresponding to a highly amphiphilic C‐terminal segment from the envelope protein (gp41) of the human immunodeficiency virus (HIV) was synthesized and tested for its ability to cause lipid membranes to fuse together (fusion) or to break open (lysis). These effects were compared to those produced by the lytic and fusogenic peptide from bee venom, melittin. Other properties studied included the changes in visible absorbance and mean particle size, and the secondary structure of peptides as judged by CD spectroscopy. Taken together, the observations suggest that protein‐mediated membrane fusion is dependent not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form an amphiphilic structure that promotes the mixing of the lipids between membranes. A speculative molecular model is proposed for membrane fusion by α‐helical peptides, and its relationship to the forces involved in protein‐membrane interactions is discussed.