bluuebunny/crossref_metadata_2025_split huggingface dataset:
Improving the binding affinity of an antibody using molecular modeling and site‐directed mutagenesis
Authors: Casipit Clayton L. , Tal Rony , Wittman Vaughan , Chavaillaz Pierre‐André , Arbuthnott Kathy , Weidanz Jon A. , Jiao Jin‐An , Wong Hing C. 
Published: February 2009

AbstractActivated Factor X releases F1.2, a 271‐amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine‐amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (KD1.22 × 10−6M). To model the TA1 antibody, we entered the sequence information of the cloned TA1Fvinto the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein fromBacillus licheniformisinEscherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.
Correlation between the 1.6 Å crystal structure and mutational analysis of keratinocyte growth factor
Authors: Osslund Timothy D. , Syed Rashid , Singer Elizabeth , Hsu Eric W.‐J. , Nybo Rebecca , Harvey Timothy , Arakawa Tsutomu , Narhi Linda Owers , Chirino Arthur , Morris Charles F. , Chen Bao‐Lu 
Published: February 2009

AbstractA comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (A23KGF‐Rl44Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a β‐trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti‐parallel β‐strands in which three pairs of the strands form a six‐stranded beta‐barrel structure and the other three pairs of β‐strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well‐defined beta strands, which form five double‐stranded anti‐parallel beta‐sheets. A sixth poorly defined β‐strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24‐33) compared to the other FGF structures, which are important for biological activity. Based on mutapenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His 127 predominantly (onfer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of Δ23KGF‐Rl44Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main‐chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic.
Binding of a substrate analog to a domain swapping protein: X‐ray structure of the complex of bovine seminal ribonuclease with uridylyl(2′,5′)adenosine
Authors: Vitagliano Luigi , Adinolfi Salvatore , Riccio Antonio , Sica Filomena , Zagari Adriana , Mazzarella Lelio 
Published: February 2009

AbstractBovine seminal ribonuclease (BS‐RNase) is a unique member of the pancreatic‐like ribonuclease superfamily. The native enzyme is a mixture of two dimeric forms with distinct structural features. The most abundant form is characterized by the swapping of N‐terminal fragments. In this paper, the crystal structure of the complex between the swapping dimer and uridylyl(2′,5′)adenosine is reported at 2.06 Å resolution. The refined model has a crystallographic R‐factor of 0.184 and good stereochemistry. The quality of the electron density maps enables the structure of both the inhibitor and active site residues to be unambiguously determined. The overall architecture of the active site is similar to that of RNase A. The dinucleotide adopts an extended conformation with the pyrimidine and purine base interacting with Thr45 and Asn71, respectively. Several residues (Gln11, Hisl2, Lys41, His119, and Phel20) bind the oxygens of the phosphate group. The structural similarity of the active sites of BS‐RNase and RNase A includes some specific water molecules believed to be relevant to catalytic activity. Upon binding of the dinucleotide, small but significant modifications of the tertiary and quaternary structure of the protein are observed. The ensuing correlation of these modifications with the catalytic activity of the enzyme is discussed.
Analysis of zinc binding sites in protein crystal structures
Authors: Alberts Ian L. , Nadassy Katalin , Wodak Shoshana J. 
Published: February 2009

AbstractThe geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H‐bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi‐zinc sites. We find that H‐bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec‐His‐Zn motif. Analysis of the stereochemistry shows that carboxyl elec‐His‐Zn motifs are geometrically rigid, while water elec‐His‐Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.
Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments
Authors: Yokota Atsushi , Takenaka Hidekazu , Oh Tomohiro , Noda Yasuo , Segawa Shin‐Ichi 
Published: February 2009

AbstractTwo peptide fragments from tuna cytochrome c (cyt c), N‐fragment (residues 1‐44 containing the heme) and C‐fragment (residues 45‐103), combine to form a 1:1 fragment complex. This was clearly proved by ion‐spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (ΔG°b, ΔHb, ΔS°b, and ΔCbp) associated with the complex formation were determined at various pHs and temperatures. ΔHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (ΔCbp) was directly determined from the temperature dependence of ΔHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/ dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.
Role of the 6‐20 disulfide bridge in the structure and activity of epidermal growth factor
Authors: Barnham Kevin J. , Torres Allan M. , Norton Raymond S. , Alewood Dianne , Alewood Paul F. , Domagala Teresa , Nice Edouard C. 
Published: February 2009

AbstractTwo synthetic analogues of murine epidermal growth factor, [Abu6, 20 mEGF4‐48 (where Abu denotes amino‐butyric acid) and [Gl, M3, K21, H40] mEGF1‐48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4‐48 was designed to determine the contribution of the 6‐20 disulfide bridge to the structure and function of mEGF. The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6‐20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N‐terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main β‐sheet from their position in native mEGF. Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4‐48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4‐48, implying that the structural changes in [Abu6, 20] mEGF4‐48, although limited to the N‐terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N‐terminal region with part of the receptor binding surface of EGF. [Gl, M3, K21, H40] mEGF1‐48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its 1H chemical shifts suggested that its structure was also very similar to native.
Probing enzyme quaternary structure by combinatorial mutagenesis and selection
Authors: Macbeath Gavin , Kast Peter , Hilvert Donald 
Published: February 2009

AbstractGenetic selection provides an effective way to obtain active catalysts from a diverse population of protein variants. We have used this tool to investigate the role of loop sequences in determining the quaternary structure of a domain‐swapped enzyme. By inserting random loops of four to seven residues into a dimeric chorismate mutase and selecting for functional variants by genetic complementation, we have obtained and characterized both monomeric and hexameric enzymes that retain considerable catalytic activity. The low percentage of active proteins recovered from these selection experiments indicates that relatively few loop sequences permit a change in quaternary structure without affecting active site structure. The results of our experiments suggest further that protein stability can be an important driving force in the evolution of oligomeric proteins.
Selecting near‐native conformations in homology modeling: The role of molecular mechanics and solvation terms
Authors: Janardhan Ajit , Vajda Sandor 
Published: February 2009

AbstractA free energy function, combining molecular mechanics energy with empirical solvation and entropic terms, is used for ranking near‐native conformations that occur in the conformational search steps of homology modeling, i.e., side‐chain search and loop closure calculations. Correlations between the free energy and RMS deviation from the X‐ray structure are established. It is shown that generally both molecular mechanics and solvation/entropic terms should be included in the potential. The identification of near‐native backbone conformations is accomplished primarily by the molecular mechanics term that becomes the dominant contribution to the free energy if the backbone is even slightly strained, as frequently occurs in loop closure calculations. Both terms become equally important if a sufficiently accurate backbone conformation is found. Finally, the selection of the best side‐chain positions for a fixed backbone is almost completely governed by the solvation term. The discriminatory power of the combined potential is demonstrated by evaluating the free energies of protein models submitted to the first meeting on Critical Assessment of techniques for protein Structure Prediction (CASP1), and comparing them to the free energies of the native conformations.
Aromatic L‐amino acid decarboxylase: Conformational change in the flexible region around arg334 is required during the transaldimination process
Authors: Ishii Seiji , Hayashi Hideyuki , Okamoto Akihiro , Kagamiyama Hiroyuki 
Published: February 2009

AbstractAromatic L‐amino acid decarboxylase (AADC) catalytic mechanism has been proposed to proceed through two consecutive intermediates (i.e., Michaelis complex and the external aldimine). Limited proteolysis of AADC that preferentially digested at the C‐terminal side of Arg334 was slightly retarded in the presence of dihydroxyphenyl acetate that formed a stable Michaelis complex. On the contrary, AADC was scarcely digested in the presence of L‐dopa methyl ester that formed a stable external aldimine. Similar protection by the substrate analogs was observed in the chemical modification experiment. From these results, we concluded that the region around Arg334 must be exposed and flexible in the unliganded state, and forming the Michaelis complex generated a subtle conformational change, then underwent marked conformational change during the subsequent transaldimination process prerequisite to forming the external aldimine.For further analyses, we constructed a mutant gene encoding in tandem the two peptides of AADC cleaved at the Asn327‐Met328 bond inside the putative flexible region. The gene product, fragmentary AADC, was still active with L‐dopa as substrate, but its kcar value was decreased 57‐fold, and the Km value was increased 9‐fold compared with those of the wild‐type AADC. The absorption spectra of the fragmentary AADC in the presence of L‐dopa methyl ester showed shift in the equilibrium of the transaldimination from the external aldimine to the Michaelis complex. Tryptic digestion of the fragmentary AADC removed seven amino acid residues, Met328‐Arg334, and resulted in complete inactivation. Susceptibility of the fragmentary enzyme to trypsin was not changed by L‐dopa methyl ester revealing the loss of appropriate conformational change in the flexible region induced by substrate binding. From these results we propose that the conformational change in the flexible region is required during the transaldimination process.
Opposite behavior of two isozymes when refolding in the presence of non‐ionic detergents
Authors: Doñate Fernando , Artigues Antonio , Iriarte Ana , Martinez‐Carrion Marino 
Published: February 2009

AbstractGroEL has a greater affinity for the mitochondrial isozyme (mAAT) of aspartate aminotransferase than for its cytosolic counterpart (cAAT) (Mattingly JR Jr, Iriarte A, Martinez‐Carrion M, 1995,J Biol Chem 270:1138‐1148), two proteins that share a high degree of sequence similarity and an almost identical spatial structure. The effect of detergents on the refolding of these large, dimeric isozymes parallels this difference in behavior. The presence of non‐ionic detergents such as Triton X‐100 or lubrol at concentrations above their critical micelle concentration (CMC) interferes with reactivation of mAAT unfolded in guanidinium chloride but increases the yield of cAAT refolding at low temperatures. The inhibitory effect of detergents on the reactivation of mAAT decreases progressively as the addition of detergents is delayed after starting the refolding reaction. The rate of disappearance of the species with affinity for binding detergents coincides with the slowest of the two rate‐limiting steps detected in the refolding pathway of mAAT. Limited proteolysis studies indicate that the overall structure of the detergent‐bound mAAT resembles that of the protein in a complex with GroEL. The mAAT folding intermediates trapped in the presence of detergents can resume reactivation either upon dilution of the detergent below its CMC or by adding β‐cyclodextrin. Thus, isolation of otherwise transient productive folding intermediates for further characterization is possible through the use of detergents.
Turn scanning by site‐directed mutagenesis: Application to the protein folding problem using the intestinal fatty acid binding protein
Authors: Kim Keehyuk , Frieden Carl 
Published: February 2009

AbstractWe have systematically mutated residues located in turns between β‐strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all β‐sheet protein exhibiting a topology comprised of two five‐stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel β‐strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C‐terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C‐terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D‐E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.
Crystallization of recombinant human heme oxygenase‐1
Authors: Schuller David J. , Poulos Thomas L. , Wilks Angela , Montellano Paul Ortiz De 
Published: February 2009

AbstractHeme oxygenase catalyzes the NADPH, 02, and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide. One of two primary isozymes, HO‐1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C‐terminus. While full‐length human HO‐1 consists of 288 residues, a truncated version with residues 1‐265 has been expressed as a soluble active enzyme in Escherichia coli. The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies. SDS gel analysis indicated that the protein had undergone proteolytic degradation. An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize. N‐terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1‐226 and 1‐237. Expression of the 1‐226 and 1‐233 versions of human HO‐1 provided active enzyme that crystallizes in a form suitable for diffraction studies. These crystals belong to space group P21, with unit cell dimensions a = 79.3 Å, b = 56.3 Å, c = 112.8 Å, and β = 101.5°.
Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories
Authors: Taylor Marc G. , Kirsch Jack F. , Rajpal Arvind 
Published: February 2009

AbstractThe rate constants, kon, for the formation of hen (chicken) lysozyme (HEWL) Fab‐ l0 complexes have been determined for wild‐type (WT) and epitope‐mutated lysozymes by a homogeneous solution method based on the 95% reduced enzymatic activity of the complex. The values fall within a narrow 10‐fold range [(0.18 to 1.92) × 106 M−1 s−1]. The affinity constants, KD, cover a broader, 440‐fold, range from 0.075 to 33 nM. Values of KD as high as 7 μM were obtained for the complexes prepared from some mutations at HEWL positions 96 and 97, but the associated kinetic constants could not be determined. The values of kon are negatively correlated with side‐chain volume at position 101HEWL, but are essentially independent of this parameter for position 21hewl substitutions. The multiple mutations made at positions 21HEWL and 101HEWL provide sufficient experimental data on complex formation to evaluate Φ values [Φ = (ΔΔG‡on)/(ΔΔGD)] at these two positions to begin to define trajectories for protein‐protein association. The data, when interpreted within the concept of a two‐step association sequence embracing a metastable encounter complex intermediate, argue that the rate determining step at position 2lHEWL (Φavg = 0.2) is encounter complex formation, but the larger Φavg value of 0.36 experienced for most position 10lHEWL mutations indicates a larger contribution from the post‐encounter annealing process at this site for these replacements.