bluuebunny/crossref_metadata_2025_split huggingface dataset:
Model for the interaction of amphiphilic helices with troponin C and calmodulin
Authors: Strynadka Natalie C. J. , James Michael N. G. 
Published: May 2005

AbstractCrystals of troponin are stabilized by an intermolecular interaction that involves the packing of helix A from the N‐terminal domain of one molecule onto the exposed hydrophobic cleft of the C‐terminal domain of a symmetry related molecules. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501–512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin‐C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1–17, 1988).
The design of idealized α/β‐barrels: Analysis of β‐sheet closure requirements
Authors: Lasters Ignace , Wodak Shoshana J. , Pio Fredric 
Published: May 2005

AbstractThe 8‐old parallel α/β‐barrel topology is encountered in proteins that display an impressive variety of functions, suggesting that this topology may be a rather nonspecific and stable folding motif. Consequently, this motif can be considered as an interesting framework to design novel proteins. It has been shown that the shape of the β‐sheet portion of the barrel can be approximated by a hyperboloid. This geometric object may therefore be used as a scaffold to construct an idealized eight‐standard β‐barrel. To facilitate the de novo design of such structures, a collection of modelling tools has been developed allowing secondary structure elements to be mapped onto the scaffold surface and rotation and translation operations to be performed about user defined axes while evaluating their contribution to the conformational energy of the system. These tools have been applied in a systematic study assessing the ϕ, ψ requirements to design symmetric eight standard β barrels with optimal hydrogen bonding between adjacent β‐strands. It is observed that: (a) the β‐sheet structure can be closed without introducing irregular stagger between β‐strands and (b) the region of ϕ, ψ dihedral angle space compatible with the formation of regular symmetric eight standard β‐barrels coincides with the ϕ, ψ region corresponding to average β‐strands in known protein structures, suggesting that barrel closure does not impose gross constraints on β‐strand geometry.
The characterization of recombinant mouse glandular kallikreins from E. coli
Authors: Blaber Michael , Isackson Paul J. , Bradshaw Ralph. A. 
Published: May 2005

AbstractA system has been developed or the expression in E. coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA. Using the epidermal growth factor binding protein (mGK‐9) and the γ‐subunit of nerve growth factor (nGK)‐3, as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated. The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste. This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factors interactions observed in this family of proteins.
Generation of potential structures for the G‐domain of chloroplast EF‐Tu using comparative molecular modeling
Authors: Lapadat Mary A. , Deerfield David W. , Pedersen Lee G. , Spremulli Linda L. 
Published: May 2005

AbstractComparative molecular modeling has been used to generate several possible structures for the G‐domain of chloroplast elongation factor Tu (EF‐Tuchl) based on the crystallographic data of the homologous E. coli protein. EF‐Tuchl contains a 10 amino acid insertion not present in the E. coli protein and this region has been modeled based on its predicted secondary structure. The insertion appears to lie on the surface of the protein. Its orientation could not be determined unequivocally but several likely structures for the nucleotide binding domain of EF‐Tuchl have been developed. The effects of the presence of water in the Mg2+ coordination sphere and of the protonation sate of the GDP ligand on the conformation of the guanine nucleotide binding site have been examined. Relative binding constants of several guanine nucleotide analogs for EF‐Tuchl have been obtained. The interactions between EF‐Tuchl and GDP predicted to be important by the models that have been developed are discussed in relation to the nucleotide binding properties of this factor and to the interactions proposed to be important in the binding of guanine nucleotides to related proteins.
Plastic adaptation toward mutations in proteins: Structural comparison of thymidylate synthases
Authors: Perry Kathy M. , Fauman Eric B. , Finer‐Moore Janet S. , Montfort William R. , Maley Gladys F. , Maley Frank , Stroud Robert M. 
Published: May 2005

AbstractThe structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 Å grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 Å resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 Å resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 Å from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.
The structure of rubredoxin from Desulfovibrio desulfuricans strain 27774 at 1.5 Å resolution
Authors: Stenkamp Ronald E. , Sieker Larry C. , Jensen Lyle H. 
Published: May 2005

AbstractThe structure of a small rubredoxin from the bacterium Desulfovibrio desulfuricans has been determined and refined at 1.5 Å resolution. The hairpin loop containing seven residues in other rubredoxins is missing in this 45 residue molecule, and once that fact was determined by amino acid sequencing studies, refinement progressed smoothly to an R value of 0.093 for all reflections from 5 to 1.5 Å resolution. Nearly all of the water molecules in the well‐ordered triclinic unit cell have been added to the crystallographic model. As in the other refined rubredoxin models, the Fe‐S4 complex is slightly distorted from ideal tetrahedral coordination.
Rate of β‐structure formation in polypeptides
Authors: Finkelstein A. V. 
Published: May 2005

AbstractAn explanation is suggested for why a marginally stable β‐structure folds extremely slowly; it is predicted that even a small increase in stability drastically accelerates β‐folding. According to the theory, this folding is a first‐order phase transition, and the rate‐limiting step is nucleation. The rate‐determining “nucleus” (transition state) is the smallest β‐sheet that is sufficiently large to provide an overall free energy reduction during subsequent folding. If the stability of the β‐structure is low, the nucleus is large and possesses a high free energy due to having a large perimeter. When the net stability of the final β‐structure increases (due to either an increase of the β‐sheet stability or a decrease in stability of the competing structures, e.g., α‐helices), the size and energy of a nucleus decrease and the rate of folding increases exponentially. This must result in a fast folding of polypeptides enriched by β‐forming residues (e.g., protein chains). The theory is developed for intramolecular β‐structure, but it can also explain the overall features of intermolecular β‐folding; it is applicable both to antiparallel and parallel β‐sheets. The difference in folding of β‐sheets, α‐helices, and proteins is discussed.
A workbench for multiple alignment construction and analysis
Authors: Schuler Gregory D. , Altschul Stephen F. , Lipman David J. 
Published: May 2005

AbstractMultiple sequence alignment can be a useful technique for studying molecular evolution, as well as for analyzing relationships between structure or function and primary sequence. We have developed for this purpose an interactive program, MACAW (Multiple Alignment Construction and Analysis Workbench), that allows the user to construct multiple alignments by locating, analyzing, editing, and combining “blocks” of aligned sequence segments. MACAW incorporates several novel features. (1) Regions of local similarity are located by a new search algorithm that avoids many of the limitations of previous techniques. (2) The statistical significance of blocks of similarity is evaluted using a recently developed mathematical theory. (3) Candidate blocks may be evaluated for potential inclusion in a multiple alignment using a variety of visualization tools. (4) A user interface permits each blocks to be edited by moving its boundaries or by eliminating particular segments, and blocks may be linked to form a composite multiple alignment. No completely automatic program is likely to deal effectively with all the complexities of the multiple alignment problem; by combining a powerful similarity search algorithm with flexible editing, analysis and display tools, MACAW allows the alignment strategy to be tailored to the problem at hand.
Comparative modeling of mammalian aspartate transcarbamylase
Authors: Scully Joshua L. , Evans David R. 
Published: May 2005

AbstractMammalian aspartate transcarbamylase (ATCase) is part of a 243 kDa multidomain polypeptide, called CAD, that catalyzes the first three steps in de novo pyrimidine biosynthesis. The structural organization of the mammalian enzyme is very different from E. coli ATCase, a dodecameric, monofunctional molecule comprised of six copies of separate catalytic and regulatory chains. Nevertheless, sequence similarities and other properties suggested that the mammalian ATCase domain and the E. coli ATCase catalytic chain have the same tertiary fold. A model of mammalian ATCase was built using the X‐ray coordinates of the E. coli catalytic chain as a tertiary template. Five small insertions and deletions could be readily accommodated in the model structure. Following energy minimization the RMS difference in the α carbon positions of the mammalian and bacterial proteins was 0.93 Å. A comparison of the hydrophobic energies, surface accessibility index, and the distribution of hydrophilic and hydrophobic residues of the CAD ATCase structure with correctly and incorrectly folded proteins and with several X‐ray structures supported the validity of the model. The mammalian ATCase domain associates to form a compact globular trimer, a prerequisite for catalysis since the active site is comprised of residues from adjacent subunits. Interactions between the clearly defined aspartate and carbamyl phosphate subdomains of the monomer were largely preserved while there was appreciable remodeling of the trimeric interfaces. Several clusters of basic residues are located on the upper surface of the domain which account in part for the elevated isoelectric point (pI = 9.4) and may represent contact regions with other more acidic domains within the chimeric polypeptide. A long interdomain linker connects the monomer at its upper surface to the remainder of the polypeptide. The configuration of active site residues is virtually identical in the mammalian and bacterial enzymes. While the CAD ATCase domain can undergo the local conformational changes that accompany catalysis in the E. coli enzyme, the high activity, closed conformation is probably more stable in the mammalian enzyme.
The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes
Authors: Noble Martin E. M. , Wierenga Rik K. , Lambeir Anne‐Marie , Opperdoes Fred R. , Thunnissen Andy‐Mark W. H. , Kalk Kor H. , Groendijk Hillie , Hol Wim G. J. 
Published: May 2005

AbstractCrystals of triosephosphate isomerase from Trypanosoma brucei brucei have been used in binding studies with three competitive inhibitors of the enzyme's activity. Highly refined structures have been deduced for the complexes between trypanosomal triosephosphate isomerase and a substrate analogue (glycerol‐3‐phosphate to 2.2 Å), a transition state analogue (3‐phosphonopropionic acid to 2.6 Å), and a compound structurally related to both (3‐phosphoglycerate to 2.2 Å). The active site structures of these complexes were compared with each other, and with two previously determined structures of triosephosphate isomerase either free from inhibitor or complexed with sulfate. The comparison reveals three conformations available to the “flexible loop” near the active site of triosephosphate isomerase: open (no ligand), almost closed (sulfate), and fully closed (phosphate/phosphonate complexes). Also seen to be sensitive to the nature of the active site ligand is the catalytic residue Glu‐167. The side chain of this residue occupies one of two discrete conformations in each of the structures so far observed. A “swung out” conformation unsuitable for catalysis is observed when sulfate, 3‐phosphoglycerate, or no ligand is bound, while a “swung in” conformation ideal for catalysis is observed in the complexes with glycerol‐3‐phosphate or 3‐phosphonopropionate. The water structure of the active site is different in all five structures. The results are discussed with respect to the triosephosphate isomerase structure function relationship, and with respect to an on‐going drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.
Free energy calculations on binding and catalysis by α‐lytic protease: The role of substrate size in the P1 pocket
Authors: Caldwell James W. , Agard David A. , Kollman Peter A. 
Published: May 2005

AbstractWe present free energy calculations using molecular dynamics on different substrates of α‐lytic protease in the gas phase, in solution, while forming a noncovalent Michaelis complex with the enzyme, and in a tetrahedral structure representing a transition state/intermediate for acylation by the enzyme. Various P1 substrates were studied, with P1 = Gly, Ala, Val, and Leu. In qualitative agreement with experiment, the enzyme was calculated to bind and catalyze most effectively substrates with P1 = Ala over those with P1 = Gly, Val or Leu. Also, the calculated relative solvation free energies of Gly → Ala and Ala → Val were in qualitative agreement with experimental values in corresponding model systems. However, the level of quantitative agreement with experiment achieved in our earlier study of relative binding and catalysis of native subtilisin and an Asn‐155 → Ala mutant was not achieved. We surmise that this is due to the greater difficulty in quantitatively simulating effects that are predominantly van der Waals and hydrophobic compared to those that are hydrogen bonding/electrostatic.
Gly‐238‐Ser substitution changes the substrate specificity of the SHV class A β‐lactamases
Authors: Lee Ki‐Young , Hopkins John D. , Syvanen Michael , O'Brien Thomas F. 
Published: May 2005

AbstractThe SHV‐type β‐lactamase SHV‐2A is related to SHV‐1 by a Gly‐238‐Ser replacement. Strains carrying SHV‐2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV‐1 are sensitive to these drugs. We present a kinetic analysis of a SHV‐1 and SHV‐2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV‐2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7‐aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV‐1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV‐2A. These results indicate that the Ser‐238‐Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV‐2A relative to SHV‐1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser‐238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV‐2A. We propose that the Gly‐238 to Ser‐238 replacement in SHV‐2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.
Protein‐protein recognition analyzed by docking simulation
Authors: Cherfils Jacqueline , Duquerroy Stéphane , Janin Joël 
Published: May 2005

AbstractAntibody–lysozyme and protease–inhibitor complexes are reconstituted by docking lysozyme as a rigid body onto the combining site of the antibodies and the inhibitors onto the active site of the proteases. Simplified protein models with one sphere per residue are subjected to simulated annealing using a crude energy function where the attractive component is proportional to the interface area. The procedure finds clusters of orientations in which a steric fit between the two protein components is achieved over a large contact surface. With five out of six complexes, the native structure of the complexes determined by X‐ray crystallography is among those retained. Docked complexes are then subjected to conformational energy refinement with full atomic detail. With Fab HyHEL 5 and lysozyme, a native‐like complex has the lowest refined energy. It can also be retrieved when starting with the X‐ray structure of free lysozyme. However, some non‐native complexes cannot be rejected: they form large interfaces, have a large number of H‐bonds, and few unpaired polar groups. While these are necessary features of protein–protein recognition, they are not sufficient in determining specificity.
Functional implications of interleukin‐1β based on the three‐dimensional structure
Authors: Veerapandian B. , Gilliland Gary L. , Raag Reetta , Svensson Anders L. , Masui Yoshihiro , Hirai Yoshikatsu , Poulos Thomas L. 
Published: May 2005

AbstractThe molecular structure of interleukin‐1β, a hormone‐like cytokine with roles in several disease processes, has been determined at 2.0 Å resolution and refined to a crystallographic R‐factor of 0.19. The frame‐work of this molecule consists of 12 antiparallel β‐strands exhibiting pseudo‐3‐fold symmetry. Six of the strands make up a β‐barrel with polar residues concentrated at either end. Analysis of the three‐dimensional structure, together with results from site‐directed mutagenesis and biochemical and immunological studies, suggest that the core of the β‐barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL‐1 interacts with its receptor.
Crystallization studies of glycosylated and unglycosylated human recombinant interleukin‐2
Authors: Stura Enrico A. , Chen Ping , Wilmot Carrie M. , Arevalo Jairo H. , Wilson Ian A. 
Published: May 2005

AbstractGlycosylated interleukin‐2 (glyIL‐2) has been crystallized in two crystal forms, and unglycosylated interleukin‐2 (uIL‐2) has been crystallized in three forms. The glycosylated form of the human recombinant IL‐2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6222 or its enantiomorph. The crystals diffract to 2.8 Å and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0–5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL‐2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.5–5.6 in the monoclinic space group P21, and less frequently in the orthorhombic space group P212121 from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross‐seeding uIL‐2 with seeds from hexagonal crystals of glyIL‐2 promotes nucleation of trigonal crystals of unglycosylated IL‐2. These trigonal crystals belong to the space group P3121 or its enantiomorph, with similar cell dimensions to the glyIL‐2 hexagonal crystals.
Metal ion stabilization of the conformation of a recombinant 19‐kDa catalytic fragment of human fibroblast collagenase
Authors: Lowry Cynthia L. , McGeehan Gerard , Le Vine Harry 
Published: May 2005

AbstractA recombinant 19‐kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full‐length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24‐nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full‐length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19‐kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 ∼60 μM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19‐kDa collagenase fragment is a multistep process stabilized by calcium.
Characterization of a putative calcium‐binding site in tobacco mosaic virus
Authors: Pattanayek Rekha , Elrod Monicia , Stubbs Gerald 
Published: May 2005

AbstractLead has been used as a substitute for calcium binding to tobacco mosaic virus (TMV). The high atomic number of lead has allowed us to use difference maps from X‐ray fiber diffraction data to characterize a calcium‐binding site in the virus. The metal ligands are slightly different from those previously believed to bind calcium to TMV, although the binding site is very close to one previously described. Two acetate groups are also bound to the lead atom. There is no significant backbone conformational change in the protein as a result of metal binding; the binding is accomplished by means of relatively small movements in amino acid side chains.