bluuebunny/crossref_metadata_2025_split huggingface dataset:
Size determination of multienzyme complexes using two‐dimensional agarose gel electrophoresis
Authors: Easom Richard A. , Debuysere Michael S. , Olson Merle S. , Serwer Philip 
Published: May 2005

AbstractIn the studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two‐dimensional agarose gel electrophoresis (2d‐AGE) (Serwer, P. et al. Anal. Biochem. 152: 339–345, 1986) was modified and employed to provide accurate sizemeasurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose inthe range of PE value needed for multienzyme complexes (effective radius, R = 10–30 nm). Appropriate conditions wereestablished to measure R value ± 1% of the pyruvate (PDC), α‐ketoglutarate (α‐KGDC), and the branched chain α‐keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy of R was limited by the accuracy of the determinations of the R value for the sizestandards. The PDC from bovine heart was found to have an R = 22.4 ± 0.2 nm following cross‐linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDCduring 2d‐AGE. All R values for the enzyme complexes studied were agreement with, though more accurate than, R valuesobtained by use of electron microscopy. In contrast to this statement, the internal dihydrolipoyl transacetylase core of PDC (E2) had an R of 18.8 ± 0.2 nm using 2d‐AGE, but 10.5 nm by electron microscopy. This observation confirms the proposal that the core of the PDC has externally projecting fibrous domains invisibleto electron microscopy.
Electrostatic effects in a large enzyme complex: Subunit interactions and electrostatic potential field of the icosahedral β60 capsid of heavy riboflavin synthase
Authors: Karshikov Andrej , Ladenstein Rudolf 
Published: May 2005

AbstractThe role of the electrostatic interactions in the stability of the icosahedral β60 capsid of heavy riboflavin synthase from Bacillus Subtilis has been investigated using an approach based on the theory of Kirkwood and Tanford. The pH dependence of the electrostatic subunit interaction agrees well with experimental data. The electrostatic subunit interaction energy has a pronounced minimum at pH 8.2 for both the ligated and ligand‐free capsid. The latter is characterized by a reduction of the magnitude and the pH range of the electrostatic attraction. It is found that only 8 charged groups, which form one cluster and two ion pairs, provide a significant contribution to the capsid stability. The analysis has shown that the aggregation/disaggregation equilibrium seems to be regulated by electrostatic interactions between β‐subunits forming dimers, which connect the relatively stable pentamers in the β‐60 capsid. The release of the ligand causesareduction of the electrostatic attraction of the dimers, which may induce disaggregation of the capsid. The electrostatic potential field due tothe titratable groups and α‐helix macrodipoles has been calculated on the basic of the Coulomb relation. Two different values of the dielectric constant have been used for the protein and the surrounding solvent, respectively. The electrostatic potential shows a radially polardistribution with a positive pole at the inner capsid wall and a negative pole outside the capsid. An interesting feature of the electrostatic field is the formation of the positive potential “channels” that coincide with the channels constituted by the pentameric and trimeric β‐subunit aggregates. It is supposed that the electrostatic potential field plays a role in enzyme‐substrate recognition.
A molecular dynamics analysis of protein structural elements
Authors: Post Carol Beth , Dobson Christopher M. , Karplus Martin 
Published: May 2005

AbstractThe relation between protein secondary structure and internal motions was examined by using molecular dynamics to calculate positional fluctuations of individual helix, β‐sheet, and loop structural elements in free and substrate‐bound hen egg‐white lysozyme. The time development of the fluctuations revealed a general correspondence between structure and dynamics; the fluctuations of the helices and β‐sheets converged within the 101 psec period of the simulation and were lower than average in magnitude, while the fluctuations of theloop regions were not converged and were mostly larger than average in magnitude. Notable exceptions to this pattern occurred in the substrate‐bound simulation. A loop region (residues 101–107) of the active site cleft had significantly reduced motion due to interactions withthe substrate. Moreover, part of a loop and a 310 helix (residues of 67–88) not in contact with the substrate showeda marked increase in fluctuations. That these differences in dynamics of free and substrate‐bound lysozyme did not result simply from sampling errors was established by an analysis of the variations in the fluctuationsof the two halves of the 101 psec simulation of free lysozyme. Concerted transitions of four to five mainchain ϕ and ψ angles between dihedral wells were shown to be responsible for large coordinate shifts in the loops. These transitions displaced six or fewer residues and took place eitherabruptly, in 1 psec or less, or with a diffusive character over 5–10 psec. Displacements of rigid secondary structures involved longer timescale motions in bound lysozyme; a 0.5 Å rms change in the position of a helix occurred over the 55 psec simulation period. This helix reorientation within the protein appears to be a response to substrate binding. There was little correlation between the solvent accessible surface areaand the dynamics of the different structural elements.
Describing protein structure: A general algorithm yielding complete helicoidal parameters and a unique overall axis
Authors: Sklenar Heinz , Etchebest Catherine , Lavery Richard 
Published: May 2005

AbstractWe present a general and mathematically rigorous algorithm which allows the helicoidal structure of a protein to be calculated starting from the atomic coordinated of its peptide backbone. This algorithm yields a unique curved axis which quantifies the folding of the backbone and a full set of helicoidal parameters describing the location of each peptide unit. The parameters obtained form a complete and independent set and can therefore be used for analyzing, comparing, or reconstructing protein backbone geometry. This algorithm has been implemented in a computer program named P‐Curve. Several examples of its possible applications are discussed.
Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm
Authors: Dohlman Jan G. , De Loof Hans , Prabhakaran M. , Koopman William J. , Segrest Jere P. 
Published: May 2005

AbstractEisenberg's helical hydrophobic moment (<μH>) algorithm was applied to the analysis of the primary structure of amphipathic α‐helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue <μH> for the amphipathic helical and control peptides were 0.46 (±/−0.07) and 0.33 (0.07) (P+0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular <μH> was plotted vs <μH>. The resulting curves are referred to as HM‐C. The mean HM‐C (of the two groups) was highly significantly different such that the HM‐C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic α helix at residues 4–21. This computer‐based method may enable rapid identification of peptide of the amphipathic α‐helix class.
Computational and site‐specific mutagenesis analyses of the asymmetric charge distribution on calmodulin
Authors: Weber P. C. , Lukas T. J. , Craig T. A. , Wilson E. , King M. M. , Kwiatkowski A. P. , Watterson D. Martin 
Published: May 2005

AbstractCalmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain α‐helices and calcium‐binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette‐based site‐specific mutagenesis of residues 82–84 or 118–120, the resulting proteins differ in their interaction with two distinct calmodulin‐dependent protein kinases, myosin light chain kinase and calmodulin‐ldependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charges in the region around residues 82–84 and 118–120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties that are important for the efficient assembly of calmodulin–protein kinase signal transduction complexes in eukaryotic cells.
Site‐directed mutagenesis of the T4 endonuclease V Gene: Mutations which enhance enzyme specific activity at low salt concentrations
Authors: Lloyd R. Stephen , Augustine Mary Lou 
Published: May 2005

AbstractPrevious structure/function analyses of the DNA repairenzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer‐specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832–1838 and 1839–1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has beeninvestigated by oligonucleotide site‐directed mutagenesis in which the codon for Tyr‐129 has been altered to reflect conservative changesof Trp and Phe and more dramatic changes of Ser‐ a stop, codon, deletion of the codon or introduction of a frameshift. Both changesto the aromatic amino acids resulted in proteins which accumulated will in E. coli and not only significantly enhanced the UV survival of repair, deficient cells but also complemented a defective denV gene within UV‐irradiated T4 phage. Partially purified preparations of the Tyr‐129 → Trp and Tyr‐129 → Phe mutants were assayed for their ability to processively incise UV‐irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr‐129 → Phe and Tyr‐129 → Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr‐129 → Phe mutant and to 20% that of wild type for the Tyr1‐29 → Trp mutant.
Hydrolysis of GTP by the α‐chain of Gs and other GTP binding proteins
Authors: Bourne Henry R. , Landis Claudia A. , Masters Susan B. 
Published: May 2005

AbstractThe functions of G proteins—like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras)—depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP‐ and GDP‐bound states. Similarities in function and amino acid sequence indicate that EF‐Tu, p21ras, and G protein α‐chains evolved from a primordial GTP‐binding protein. Proteins in all three families appear to share common mechanisms for GTP‐dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the α‐chain of Gs (αs) point to key regions that are involved in GTP‐dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of αs in human pituitary tumors inhibit‐the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in αs that corresponds to Gln‐61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of the GTPase inhibiting mutations in αs occurs in the codon for an ARG residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by αs. This Arg residue is located in a domain of αs not represented in EF‐Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF‐Tu by the programmed ribosome and for p21ras by the recently discovered GTPase‐activating protein. Owing to their inherited similarities of structure and function, what we learn about αs, p21ras, or EF‐tu as individual molecules helps us to understand crucial functions of other members of the super‐family.
The carboxyl terminal domain of Escherichia coli DNA topoisomerase I confers higher affinity to DNA
Authors: Beran‐Steed Rita K. , Tse‐Dinh Yuk‐Ching 
Published: May 2005

AbstractLimited digestion of E. coli DNA topoisomerase I with trypsin or papain generated a DNA‐binding domain of MW 14,000 corresponding to the carboxyl terminal of the enzyme. This fragment binds to single‐stranded DNA agarose as tightly as the intact enzyme. It required around 400 mM NaCl for elution. A truncated topoisomerase that lacks this C‐terminal domain was purified. It was eluted from the single‐stranded DNA agarose column at around 150 mM NaCl. Although the truncated enzyme could relax negatively supercoiled DNA as efficiently as the intact enzyme at low ionic strength, its processivity was more sensitive to increasing salt concentration. Measurement of binding to fluorescent etheno‐M13 DNA also demonstrated that the presence of the C‐terminal domain confers higher affinity to DNA for the enzyme.
A hydrophobic cluster forms early in the folding of dihydrofolate reductase
Authors: Garvey E. P. , Swank J. , Matthews C. R. 
Published: May 2005

AbstractThe rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase from Escherichia coli was examined by site‐directed mutagenesis and by physicochemical means. The absence of this fluorescence‐detected phase in the refolding of the Trp‐74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp‐74. In addition, water‐soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and nativelike conformers. These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp‐74 fromsolvent.
The interaction of calmodulin with fluorescent and photoreactive model peptides: Evidence for a short interdomain separation
Authors: O'Neil K. T. , DeGrado William F. 
Published: May 2005

AbstractCalmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2+‐dependent manner. Recently, we introduced a photoaffinity label, p‐benzoylphenylalanine (Bpa), into the sequence of a model, α‐helical, calmodulin‐binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met‐144 on the C‐terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met‐71 on the N‐terminal do main was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long α‐helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin‐binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross‐link between Met‐71 and Met 124 on the N‐ and C‐terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthsized. After photocross‐linking the Bpa redidue of this peptide to Met‐71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C‐terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphi peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal sturcture of the uncommplexed protein.
The neuraminidase of influenza virus
Authors: Air Gillian M. , Laver W. Graeme 
Published: May 2005

AbstractIt is the enzyme neuraminidase, projecting form the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3‐dimensional structure of the epitopes was obtained by X‐ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.
Acid‐induced dimerization of skeletal troponin C
Authors: Wang Chien‐Kao , Lebowitz Jacob , Cheung Herbert C. 
Published: May 2005

AbstractWe have investigated pH‐dependent changes of the properties of troponin C from rabbit skeletal muscle. At pH 7.5 this protein is a monomer and at pH 5.2 it is a dimer. In contrast, bovine cardiac troponin C remains essentially monomeric at pH 5.2. Bovine brain calmodulin is not a dimer, but significantly aggregated at the same acidic pH. The dimerization of skeletal troponin C was demonstrated by low‐speed (16,000 rpm) sedimentation equilibrium measurements carried out at 20°C and by polyacrylamide gel electrophoresis under nondenaturing conditions. Dimer formation was significantly inhibited in the ultracentrifuge at rotor speeds of 30,000 and 40,000 rpm at 20°C, and was completely prevented at a rotor speed of 40,000 rpm and 4°C. This temperature and pressure dependence of dimerization strongly suggests that hydrophobic bonding is a major factor in promoting skeletal troponin C association at pH 5.2. The intramolecular distance between Met‐25 and Cys‐98 of rabbit skeletal troponin C deduced from fluorescence resonance energy transfer measurements increased by a factor of two upon lowering the pH from 7.5 to 5.2, indicating a pH‐dependent transition in which the protein changed from a relatively compact conformation to an elongated conformation. The protein‐induced increase in the energy transfer distance is related to the acid‐induced dimerization of the protein. The extended conformation observed at pH 5.2 is compatible with the dumbbell‐shaped structure of skeletal troponin C crystals obtained from turkey at pH 5.0 [Herzberg, O., James, M. N. G. Nature (London) 313:653–659, 1985] and chicken at pH 5.1 (Sundaralingam, M., Bergstrome, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., Wang, B. C. Science 227:945–948, 1985). However, the conformation in neural solution deviates form that predicted by crystallography. Intermolecular interactions leading to dimer formation likely play an important role in promoting the extended conformation that exists at acidic pH.
A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics
Authors: Coplen Lonnie J. , Frieden Richard W. , Goldenberg David P. 
Published: May 2005

AbstractA genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of wild‐type protein has a half‐time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 “DTT‐sensitive” mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the naive wild‐type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated a most sites without completely preventing folding Because this genetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.