bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chymotrypsin inhibitory activity of normal C1‐inhibitor and a P1 Arg to His mutant: Evidence for the presence of overlapping reactive centers
Authors: Aulak K.S. , Davis A.E. , Donaldson V.H. , Harrison R.A. 
Published: February 2009

AbstractC1‐inhibitor is a serine proteinase inhibitor that is active against C1s, C1r, kallikrein, and factor XII. Recently, it has been shown that it also has inhibitory activity against chymotrypsin. We have investigated this activity of normal human C1‐inhibitor, normal rabbit C1‐inhibitor, and P1 Arg to His mutant human C1‐inhibitors and find that all are able to inhibit chymotrypsin and form stable sodium dodecyl sulfate‐resistant complexes. The Kass values show that the P1 His mutant is a slightly better inhibitor of chymotrypsin than normal human C1‐inhibitor (3.4 × 104 compared with 7.3 × 103). The carboxy‐terminal peptide of normal human C1‐inhibitor, derived from the dissociated protease‐inhibitor complex, shows cleavage between the P2 and P1 residues. Therefore, as with α2‐antiplasmin, C1‐inhibitor possesses two overlapping P1 residues, one for chymotrypsin and the other for Arg‐specific proteinases. In contrast, with the P1 His mutant, the peptide generated from the dissociation of its complex with chymotrypsin demonstrated cleavage between the P1 and P'1 residues. Therefore, unlike α2‐antiplasmin, chymotrypsin utilizes the P2 residue as its reactive site in normal C1‐inhibitor but utilizes the P1 residue as its reactive site in the P1 His mutant protein. This suggests that the reactive center loop allows a degree of induced fit and therefore must be relatively flexible.
Crystal structure analysis of amicyanin and apoamicyanin from paracoccus denitrificans at 2.0 Å and 1.8 Å resolution
Authors: Durley Rosemary , Chen Longyin , Scott Mathews F. , Lim Louis W. , Davidson Victor L. 
Published: February 2009

AbstractThe crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 Å resolution using energy‐restrained least‐squares procedures to a crystallographic residual of 15.7%. The copper‐free protein, apoamicyanin, has also been refined to 1.8 Å resolution with residual 15.5%.The protein is found to have a β‐sandwich topology with nine β‐strands forming two mixed β‐sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N‐terminus that have no equivalents in the other proteins; a portion of these residues forms the first β‐strand of the structure.The copper atom is located in a pocket between the β‐sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between β‐strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper.One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.
Binding of amino acid side chains to preformed cavities: Interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues
Authors: Bigler Theresa L. , Lu Wuyuan , Park Soon Jae , Tashiro Misao , Wieczorek Maciej , Wynn Richard , Laskowski Michael 
Published: February 2009

AbstractIn the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine α chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight‐chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side‐chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side‐chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. β‐Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of γ‐branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains.Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6–18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19–56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive‐site peptide bond X18‐Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive‐site‐intact inhibitor variants.
Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from thermus thermophilus: Differences in dimer–dimer interaction
Authors: Wagner Ulrike G. , Frolow Felix , Sussman Joel L. , Pattridge Katherine A. , Ludwig Martha L. , Stallings William C. , Werber Moshe M. , Oefner Christian 
Published: February 2009

AbstractThe three‐dimensional X‐ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P21212 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787–788). Refinement of the protein structure (3, 148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R‐factor of 0.207 using all data from 8.0 to 3.2 Å resolution and group thermal parameters. The monomer–monomer interactions typical of bacterial Fe‐ and Mn‐containing SODs are retained in the human enzyme, but the dimer–dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84° relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer–dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal—ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.
Stress and strain in staphylococcal nuclease
Authors: Hodel Alec , Kautz Roger A. , Jacobs Marc D. , Fox Robert O. 
Published: February 2009

AbstractProtein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well‐refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIaβ‐turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116‐Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X‐ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116–117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112–117), which contains a predominantly trans Gly 116–Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the β‐carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112–117 loop only allow trans conformations where the local backbone interactions associated with the ϕ and Ψ torsion angles are strained. When the 116–117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X‐Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.
The molten globule intermediate of apomyoglobin and the process of protein folding
Authors: Barrick Doug , Baldwin Robert L. 
Published: February 2009

AbstractThe molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of α‐lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87–103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen‐deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid‐induced unfolding transition, which have been determined by fitting a minimal three‐state model (N ↔ I ↔ U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two‐dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544–1548). Directed mutagenesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113–4118), which can explain why I is formed rapidly from U at the start of folding. These experiments are consistent with the explanation proposed earlier (Baldwin, R.L., 1989, Trends Biochem. Sci. 14, 291–294) for the stabilization of native secondary structures in molten globule intermediates, namely that each unit of native secondary structure has a hydrophobic face, and the hydrophobic surfaces of two units of secondary structure can interact loosely to provide mutual stabilization. These topics are discussed here in the light of some additional results.
Families and the structural relatedness among globular proteins
Authors: Yee David P. , Dill Ken A. 
Published: February 2009

AbstractProtein structures come in families. Are families “closely knit” or “loosely knit” entities? We describe a measure of relatedness among polymer conformations. Based on weighted distance maps, this measure differs from existing measures mainly in two respects: (1) it is computationally fast, and (2) it can compare any two proteins, regardless of their relative chain lengths or degree of similarity. It does not require finding relative alignments. The measure is used here to determine the dissimilarities between all 12, 403 possible pairs of 158 diverse protein structures from the Brookhaven Protein Data Bank (PDB). Combined with minimal spanning trees and hierarchical clustering methods, this measure is used to define structural families. It is also useful for rapidly searching a dataset of protein structures for specific substructural motifs. By using an analogy to distributions of Euclidean distances, we find that protein families are not tightly knit entities.
Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni
Authors: Middaugh C. Russell , Ryan James A. , Burke Carl J. , Mach Henryk , Naylor Adel M. , Bogusky Michael J. , Pitzenberger Steven M. , Ji Hanlee , Cordingley John S. , Thomson John A. 
Published: February 2009

AbstractThe peptide (Gly‐l‐Tyr‐l‐Asp‐l‐Lys‐l‐Tyr)6, referred to as F4–6, was synthesized as a model for a schistosome eggshell protein in which the Gly‐Tyr‐Asp‐Lys‐Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotometric titrations, NMR, and molecular modeling suggests that F4–6 forms some type of left‐handed structure. A likely possibility appears to be a left‐handed α‐helix stabilized by Lysi‐Aspi+4 salt bridges and possibly Aspi‐Tyri+4 hydrogen bonding and Tyr‐Tyr interactions. Spectroscopic studies of a number of F4–6 analogues support this conclusion. For example, substitution of d‐Ala for Gly produces a peptide with enhanced left‐handed helical spectral characteristics, whereas an l‐Ala substitution results in a peptide with minimal structure. These studies suggest that the F4 protein from Schistosoma mansoni may be the first example of a naturally occurring protein devoid of proline and carbohydrate that forms a left‐handed helix composed of l‐amino acids, although alternative forms of other left‐handed structures have yet to be rigorously excluded.
Brownian dynamics simulations of molecular recognition in an antibody‐antigen system
Authors: Kozack Richard E. , Subramaniam Shankar 
Published: February 2009

AbstractThe crystal structure for an antibody‐antigen system, that of the anti‐hen egg lysozyme monoclonal antibody HyHEL‐5 complexed to lysozyme, is used as the starting point for computer simulations of diffusional encounters between the two proteins. The investigation consists of two parts: first, the linearized Poisson‐Boltzmann equation is solved to determine the long‐range electrostatic forces between antibody and antigen, and then, the relative motion as influenced by these forces is modeled within Brownian motion theory. The effects of various point mutations on the calculated reaction rate are considered. It is found that charged residues close to the binding site exert the greatest influence in steering the proteins into a configuration favorable for their binding, while more distant mutations are qualitatively described by the Smoluchowski model for the mutual diffusion of two uniformly charged spheres. The antibody residues involved in forming salt links with the lysozyme, Glu‐H35 and Glu‐H50, appear to be particularly important in electrostatic steering, as neutralization of both of them yields reaction rates that are two to three orders of magnitude below those of wild‐type rates. The relative rates obtained from the simulations can be tested through kinetic measurements on mutant protein complexes. Kinetically efficient partners can also be designed and constructed through directed mutagenesis.
Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: Evidence for an uncoupled R state
Authors: Tanner John J. , Smith Paul E. , Krause Kurt L. 
Published: February 2009

AbstractIn the R form of ATCase complexed with the bisubstrate analogue, N‐(phosphonacetyl)‐l‐aspartate, large temperature factors are reported for the allosteric domains of the regulatory chains. We studied the conformational flexibility of the holoenzyme with molecular dynamics simulations and rigid body (TLS) analysis. The results of the molecular dynamics simulations suggest that, although local atomic fluctuations account for the temperature factors of the catalytic and zinc domains, they do not account for the large temperature factors of the allosteric regions. However, the temperature factors of the allosteric domains can be satisfactorily analyzed using a rigid body model. The simulations and rigid body analysis support the idea that the allosteric regions are mechanically uncoupled from the rest of the enzyme in the PALA structure. Implications of this uncoupling for allosteric regulation are discussed.
Structures of DNA‐binding mutant zinc finger domains: Implications for DNA binding
Authors: Hoffman Ross C. , Klevit Rachel E. , Horvath Suzanna J. 
Published: February 2009

AbstractStudies of Cys2‐His2 zinc finger domains have revealed that the structures of individual finger domains in solution determined by NMR spectroscopy are strikingly similar to the structure of fingers bound to DNA determined by X‐ray diffraction. Therefore, detailed structural analyses of single finger domains that contain amino acid substitutions known to affect DNA binding in the whole protein can yield information concerning the structural ramifications of such mutations. We have used this approach to study two mutants in the N‐terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2‐His2 zinc finger sequences spanning residues 102‐159. Two point mutants at position 118 in the N‐terminal zinc finger (ADR1b: 102‐130) that adversely affect the DNA‐binding activity of ADR1 have previously been identified: H118A and H118Y. The structures of wild‐type ADR1b and the two mutant zinc finger domains were determined using two‐dimensional nuclear magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated. The molecular architecture of the refined wild‐type ADR1b domain is presented in detail. Comparisons of wild‐type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild‐type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2‐His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein‐DNA interaction.
Disulfide bonds in homo‐ and heterodimers of EF‐hand subdomains of calbindin D9k: Stability, calcium binding, and NMR studies
Authors: Linse Sara , Thulin Eva , Sellers Peter 
Published: February 2009

AbstractThe effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39 → Cys, Ile 73 → Cys, and Pro 43 → Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 °C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF‐hand subdomains are equally stable regardless if the covalent link is the 43‐44 peptide bond or the disulfide bond.Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF‐hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive cooperativity, and an average affinity of 106 M−1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C‐terminal fragment Tm increases from 59 + 2 without covalent linkage to 91 + 2 °C with disulfide, and Cm from = 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.
In vivo formation of active aspartate transcarbamoylase from complementing fragments of the catalytic polypeptide chains
Authors: Yang Ying R. , Schachman H. K. 
Published: February 2009

AbstractDespite the complexity of Escherichia coli aspartate transcarbamoylase (ATCase), composed of 12 polypeptide chains organized as two catalytic (C) trimers and three regulatory (R) dimers, it is possible to form active stable enzyme in vivo even with fragmented catalytic (c) chains. Based on the observation that chymotryptic digestion of the C trimers yields an active protein that can be dissociated into fragmented chains and then reconstituted in high yield, genetically engineered plasmids carrying the genes encoding each of the fragments were constructed. When the N‐terminal peptide (residues 1‐242) and the C‐terminal peptide (residues 235‐310) were expressed separately, each incomplete polypeptide chain was found in the insoluble fraction of the individual cell extracts. Mixing the two insoluble pellets in 6.5 M urea, followed by a 10‐fold dilution in buffer, led to the formation of active C trimers composed of incomplete polypeptide chains with an 8‐amino acid redundancy. When the two partial genes were linked into a single transcriptional unit separated by a 15‐nucleotide untranslated region containing a sequence for ribosome binding, the cells produced high yields of active C trimers composed of the incomplete, partially overlapping chains. The resulting protein, purified as C trimers or as holoenzyme formed by the addition of R subunits, has a specific activity (Vmax) only slightly less than that of the wild‐type C trimer and ATCase. However, Km for aspartate exhibited by the C trimer composed of fragmented chains is more than 10‐fold larger than that of the wild‐type trimer. The holoenzyme formed from the C trimer containing the coexpressed peptides is devoid of cooperativity with a Hill coefficient of 1.0, as contrasted to wild‐type ATCase for which the Hill coefficient is 1.7. Km for aspartate as well as Kd for the binding of the bisubstrate analog N‐(phosphonacetyl)‐l‐aspartate are significantly higher than the analogous values for wild‐type ATCase. Sedimentation velocity experiments indicate that the holoenzyme containing the incomplete chains has a conformation analogous to that of the R state of wild‐type ATCase.