bluuebunny/crossref_metadata_2025_split huggingface dataset:
cAMP‐dependent protein kinase: Crystallographic insights into substrate recognition and phosphotransfer
Authors: Madhusudan  , Trafny Elzbieta A. , Xuong Nguyen‐Huu , Adams Joseph A. , Eyck Lynn F.Ten , Taylor Susan S. , Sowadski Janusz M. 
Published: July 2010

AbstractThe crystal structure of ternary and binary substrate complexes of the catalytic subunit of CAMP‐dependent protein kinase has been refined at 2.2 and 2.25 Å resolution, respectively. The ternary complex contains ADP and a 20‐residue substrate peptide, whereas the binary complex contains the phosphorylated substrate peptide. These 2 structures were refined to crystallographic R‐factors of 17.5 and 18.1%, respectively. In the ternary complex, the hydroxyl oxygen OG of the serine at the P‐site is 2.7 Å from the OD1 atom of Asp 166. This is the first crystallographic evidence showing the direct interaction of this invariant carboxylate with a peptide substrate, and supports the predicted role of Asp 166 as a catalytic base and as an agent to position the serine ‐OH for nucleophilic attack. A comparison of the substrate and inhibitor ternary complexes places the hydroxyl oxygen of the serine 2.7 Å from the γ‐phosphate of ATP and supports a direct in‐line mechanism for phosphotransfer. In the binary complex, the phosphate on the Ser interacts directly with the 6N of Lys 168, another conserved residue. In the ternary complex containing ATP and the inhibitor peptide, Lys 168 interacts electrostatically with the γ‐phosphate of ATP (Zheng J, Knighton DR, Ten Eyck LF, Karlsson R, Xuong NH, Taylor SS, Sowadski JM, 1993, Biochemistry 32:2154‐2161). Thus, Lys 168 remains closely associated with the phosphate in both complexes. A comparison of this binary complex structure with the recently solved structure of the ternary complex containing ATP and inhibitor peptide also reveals that the phosphate atom traverses a distance of about 1.5 Å following nucleophilic attack by serine and transfer to the peptide. No major conformational changes of active site residues are seen when the substrate and product complexes are compared, although the binary complex with the phosphopeptide reveals localized changes in conformation in the region corresponding to the glycine‐rich loop. The high B‐factors for this loop support the conclusion that this structural motif is a highly mobile segment of the protein.
Sculpting proteins interactively: Continual energy minimization embedded in a graphical modeling system
Authors: Surles Mark C. , Richardson Jane S. , Richardson David C. , Brooks Frederick P. 
Published: July 2010

AbstractWe describe a new paradigm for modeling proteins in interactive computer graphics systems — continual maintenance of a physically valid representation, combined with direct user control and visualization. This is achieved by a fast algorithm for energy minimization, capable of real‐time performance on all atoms of a small protein, plus graphically specified user tugs. The modeling system, called Sculpt, rigidly constrains bond lengths, bond angles, and planar groups (similar to existing interactive modeling programs), while it applies elastic restraints to minimize the potential energy due to torsions, hydrogen bonds, and van der Waals and electrostatic4 interactions (similar to existing batch minimization programs), and user‐specified springs. The graphical interface can show bad and/or favorable contacts, and individual energy terms can be turned on or off to determine their effects and interactions.Sculpt finds a local minimum of the total energy that satisfies all the constraints using an augmented Lagrange‐multiplier method; calculation time increases only linearly with the number of atoms because the matrix of constraint gradients is sparse and banded. On a 100‐MHz MIPS R4000 processor (Silicon Graphics Indigo), Sculpt achieves 11 updates per second on a 20‐residue fragment and 2 updates per second on an 80‐residue protein, using all atoms except non‐H‐bonding hydrogens, and without electrostatic interactions. Applications of Sculpt are described: to reverse the direction of bundle packing in a designed 4‐helix bundle protein, to fold up a 2‐stranded β‐ribbon into an approximate β‐barrel, and to design the sequence and conformation of a 30‐residue peptide that mimics one partner of a protein subunit interaction.Computer models that are both interactive and physically realistic (within the limitations of a given force field) have 2 significant advantages: (1) they make feasible the modeling of very large changes (such as needed for de novo design), and (2) they help the user understand how different energy terms interact to stabilize a given conformation. The Sculpt paradigm combines many of the best features of interactive graphical modeling, energy minimization, and actual physical models, and we propose it as an especially productive way to use current and future increases in computer speed.
β‐Sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins
Authors: Wen Z. Q. , Hecht L. , Barron L. D. 
Published: July 2010

AbstractWe have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, α‐chymotrypsin, and β‐lactoglobulin, all of which are rich in β‐sheet, together with that of the model β‐turn peptide L‐pro‐L‐leu‐gly‐NH2. Possible ROA signatures of antiparallel β‐sheet include a strong sharp positive band at ∼ 1,313 cm” associated with backbone amide III CαH and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at ∼1,658 cm−1. Negative ROA bands in the range ∼1,340‐1,380 cm−1, which might originate in glycine CH2 deformations, appear to be characteristic of β‐turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.
Enlarged representative set of protein structures
Authors: Hobohm Uwe , Sander Chris 
Published: July 2010

AbstractTO reduce redundancy in the Protein Data Bank of 3D protein structures, which is caused by many homologous proteins in the data bank, we have selected a representative set of structures. The selection algorithm was designed to (1) select as many nonhomologous structures as possible, and (2) to select structures of good quality. The representative set may reduce time and effort in statistical analyses.
DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*)
Authors: Rodgers Karla K. , Coleman Joseph E. 
Published: July 2010

AbstractThe DNA binding domain of the GAL4 transcription factor from yeast is located in the N‐terminal 60 residues of the polypeptide of 881 amino acids. This domain binds 2 Zn ions, which form a binuclear cluster, Zn2C6 with 6 C residues, two of which bridge the 2 metal ions (Gardner KH et al., 1991, Biochemistry 30:11292‐11302). Binding of Zn or Cd to GAL4 induces the conformation of the protein necessary to recognize the specific DNA sequence, UASG, to which GAL4 binds as a dimer. Gel retardation assays have been utilized to determine the relative affinities of the Zn2 and Zn1 forms of the N‐terminal 149 residues of GAL4, GAL4(149*), for UASG DNA sequences. We show that Cd2‐ and Zn1 GAL4(149*) bind to UASG DNA with 2‐fold and 4‐8‐fold lower affinities than Zn2GAL4(149*), respectively. Thus, the metal species and the number of metal ions bound have measurable effects on the specific DNA binding affinity of GAL4, but these differences are small in comparison to the ratio, >103 under some conditions, that characterizes the specific to nonspecific DNA binding affinities of the N‐terminal fragments of GAL4. A shorter N‐terminal fragment, GAL4(62*), although it continues to recognize the UASG sequence with a high degree of specificity, binds with 1,000‐2,000‐fold lower affinity than does Zn2GAL4(149*). Gel retardation titrations of a DNA containing 2 UASG sites with increasing concentrations of GAL4(62*) generate a series of 4 retarded bands in contrast to 2 retarded bands formed when the the same DNA is titrated with GAL4(149*). These data suggest that GAL4(62*) binds to the UASG sites as individual monomers that dimerize on the DNA, whereas GAL4(149*) binds the UASG DNA cooperatively as a dimer. The > 103 lower affinity of GAL4(62*) for the UASG DNA can be accounted for by its failure to form dimers in solution. Zn2‐, Zn1‐, or Cd2GAL4(149*) induces differential rates of gel migration in a series of circularly permutated UASG‐containing DNA restriction fragments. Analysis of the data suggests that all 3 proteins cause a 26° angle of bend in the DNA when bound to 1 UASG site and 45° when bound to 2 tandem UASG sites. The same assay shows that GAL4(62*) does not induce significant bending of the UASG DNA sequences. Thus, the additional subdomains found in the larger polypeptide fragment, GAL4(149*), must exert an additional force on the DNA either through direct contacts with the DNA or indirectly through altered protein conformation.
Slow‐folding kinetics of ribonuclease‐A by volume change and circular dichroism: Evidence for two independent reactions
Authors: Ybe Joel A. , Kahn Peter C. 
Published: July 2010

AbstractThe slow refolding of guanidine‐HC1‐denatured ribonuclease‐A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of ‐64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the “XY” reaction of Lin and Brandts (1983, Biochemistry 22:563‐573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the “XY” process. The early formation of a compact structure in the event responsible for the rapid +232‐mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157‐6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124‐6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.
Retrospective analysis of a secondary structure prediction: The catalytic domain of matrix metalloproteinases
Authors: Hodgkin Edward E. , Gillman Ian C. , Gilbert Richard J. 
Published: July 2010

AbstractSecondary structure prediction of the catalytic domain of matrix metalloproteinases is evaluated in the light of recently published experimentally determined structures. The prediction was made by combining conformational propensity, surface probability, and residue conservation calculated for an alignment of 19 sequences. The position of each observed secondary structure element was correctly predicted with a high degree of accuracy, with a single β‐strand falsely predicted. The domain fold was also anticipated from the prediction by analogy with the structural elements found in the distantly related metalloproteinases thermolysin, astacin, and adamalysin.
A recombinant human hemoglobin with asparagine‐102(β) substituted by alanine has a limiting low oxygen affinity, reduced marginally by chloride
Authors: Yanase Hideshi , Manning Lois R. , Vandegriff Kim , Winslow Robert M. , Manning James M. 
Published: July 2010

AbstractA recombinant (r) mutant hemoglobin (Hb) with Asn‐102(β) replaced by an Ala (N102A(β)) has been prepared by PCR amplification of a mutagenic DNA fragment and expression of the recombinant protein in yeast. The side chain of Asn‐102(β) is part of an important region of the α1β2 interface that undergoes large structural changes in the transition between the deoxy and oxy conformations. Three natural mutant Hbs with neutral substitutions of Thr, Ser, or Tyr at this site have low oxygen affinities because a hydrogen bond between Asn‐102(β) and Asp‐94(α) in normal HbA was considered to be absent in these mutants, thereby destabilizing the oxy conformation in favor of the deoxy conformation. This proposal has been tested by expression of an rHb containing alanine at position 102(0); alanine was chosen because its methyl side chain cannot participate in hydrogen bond formation, yet it is small enough not to disrupt the subunit interface. The nature of the desired replacement was established by sequencing the entire mutated β‐globin gene as well as the tryptic peptide containing the substitution. Further characterization by SDS‐PAGE, isoelectric focusing, HPLC analysis, mass spectrometry, amino acid analysis, and sequencing of the mutant tryptic peptide confirmed the purity of the rHb. Its oxygen binding curve (2.4 mM in heme) in the absence of chloride showed that it had a very low oxygen affinity with a P50 of 42 mm Hg. In the presence of added chloride (0.5 M), its oxygen affinity was further reduced only slightly to a P50 of 49 mm Hg. In contrast, the oxygen affinity of HbA was lowered two‐ to threefold by the same concentration of chloride. Comparison of the properties of the rHb N102A(β) with those of the rHb with Asp‐99(β), which is at this same subunit interface, substituted by a Lys (Yanase H, et al., 1994, Protein Sci 3:1213–1223), demonstrates how the judicious choice of the amino acid substitution based upon the properties of natural mutations at a particular site can further enhance our understanding of the role of certain amino acid side chains in Hb function.
A procedure for the automatic determination of hydrophobic cores in protein structures
Authors: Swindells Mark B. 
Published: July 2010

AbstractAn algorithm is described for automatically detecting hydrophobic cores in proteins of known structure. Three pieces of information are considered in order to achieve this goal. These are: secondary structure, side‐chain accessibility, and side‐chain‐side‐chain contacts. Residues are considered to contribute to a core when they occur in regular secondary structure and have buried side chains that form predominantly nonpolar contacts with one another. This paper describes the algorithm's application to families of proteins with conserved topologies but low sequence similarities. The aim of this investigation is to determine the efficacy of the algorithm as well as to study the extent to which similar cores are identified within a common topology.
Self‐organizing hierarchic networks for pattern recognition in protein sequence
Authors: Hanke Jens , Beckmann Georg , Bork Peer , Reich Jens G. 
Published: July 2010

AbstractWe present a method based on hierarchical self‐organizing maps (SOMs) for recognizing patterns in protein sequences. The method is fully automatic, does not require prealigned sequences, is insensitive to redundancy in the training set, and works surprisingly well even with small learning sets. Because it uses unsupervised neural networks, it is able to extract patterns that are not present in all of the unaligned sequences of the learning set. The identification of these patterns in sequence databases is sensitive and efficient.The procedure comprises three main training stages. In the first stage, one SOM is trained to extract common features from the set of unaligned learning sequences. A feature is a number of ungapped sequence segments (usually 4–16 residues long) that are similar to segments in most of the sequences of the learning set according to an initial similarity matrix. In the second training stage, the recognition of each individual feature is refined by selecting an optimal weighting matrix out of a variety of existing amino acid similarity matrices. In a third stage of the SOM procedure, the position of the features in the individual sequences is learned. This allows for variants with feature repeats and feature shuffling.The procedure has been successfully applied to a number of notoriously difficult cases with distinct recognition problems: helix‐turn‐helix motifs in DNA‐binding proteins, the CUB domain of developmentally regulated proteins, and the superfamily of ribokinases. A comparison with the established database search procedure PROFILE (and with several others) led to the conclusion that the new automatic method performs satisfactorily.
Conformation of the hypervariable region L3 without the key proline residue
Authors: Guarné Alba , Bravo Jerónimo , Calvo Javier , Lozano Francisco , Vives Jordi , Fita Ignasi 
Published: July 2010

AbstractThe refined structure of the Fab fragment of the monoclonal antibody CRIS‐I (IgG2ak) against the leukocyte differentiation antigen CD5, determined at 1.9 Å resolution with an agreement R‐factor of 18.3%, reveals a variant of the canonical conformations proposed for the light chain complementarity determining region L3 (CDR‐L3). This is the first Fab structure available with a K light chain in which the CDR‐L3 lacks the key proline residue in either position 94 or 95. The conformation found could be significant for about 10% of the murine IgG molecules with k light chains without proline in their CDR‐L3 sequences.
Structure‐based modeling of the ligand binding domain of the human cell surface receptor CD23 and comparison of two independently derived molecular models
Authors: Bajorath Jürgen , Aruffo Alejandro 
Published: July 2010

AbstractCD23, a type II membrane receptor protein, recognizes four different ligands via its extracellular C‐type lectin domain: immunoglobulin E (IgE), CD21, and the β2‐integrins CD11b and CD11c. CD23 specifically interacts in a calcium‐dependent manner, “lectin‐like” with carbohydrate moieties expressed on CD21 and CD11b/c, but also “lectin‐unlike” with protein epitopes on IgE. As a first step in analyzing the multiple binding specificities associated with CD23 in more detail, we report a detailed molecular model of the lectin‐like domain of human CD23 (hCD23). The model was built based on information provided by X‐ray structures of mannose binding protein (MBP) and E‐selectin, both of which are members of the calcium‐dependent (C‐type) lectin superfamily. Sequence‐structure comparisons suggest that hCD23 is structurally more similar to MBP than to E‐selectin. The hCD23 model is compared to an independently derived model. Although the CD23‐carbohydrate and CD23‐protein interactions are both calcium dependent, analysis of the model suggests the presence of distinct binding sites for these ligands.
Phosphorylation‐induced torsion‐angle strain in the active center of HPr, detected by NMR and restrained molecular dynamics refinement
Authors: Van Nuland Nico A. J. , Wiersma Jonna A. , Spoel David Van Der , De Groot Bert L. , Scheek Ruud M. , Robillard George T. 
Published: July 2010

AbstractThe structure of the phosphorylated form of the histidine‐containing phosphocarrier protein HPr from Escherichia coli has been solved by NMR and compared with that of unphosphorylated HPr. The structural changes that occur upon phosphorylation of His 15, monitored by changes in NOE patterns, 3JNHHα‐coupling constants, and chemical shifts, are limited to the region around the phosphorylation site. The His 15 backbone torsion angles become strained upon phosphorylation. The release of this strain during the phosphoryl‐transfer to Enzyme II facilitates the transport of carbohydrates across the membrane.From an X‐ray study of Streptococcus faecalis HPr (Jia Z, Vandonselaar M, Quail JW, Delbaere LTJ, 1993, Nature 361:94–97), it was proposed that the observed torsion‐angle strain at residue 16 in unphosphorylated S. faecalis HPr has a role to play in the protein's phosphocarrier function. The model predicts that this strain is released upon phosphorylation. Our observations on E. coli HPr in solution, which shows strain only after phosphorylation, and the fact that all other HPrs studied thus far in their unphosphorylated forms show no strain either, led us to investigate the possibility that the crystal environment causes the strain in S. faecalis HPr. A 1‐ns molecular dynamics simulation of S. faecalis HPr, under conditions that mimic the crystal environment, confirms the observations from the X‐ray study, including the torsion‐angle strain at residue 16. The strain disappeared, however, when S. faecalis HPr was simulated in a water environment, resulting in an active site configuration virtually the same as that observed in all other unphosphorylated HPrs. This indicates that the torsion‐angle strain at Ala 16 in S. faecalis HPr is a result of crystal contacts or conditions and does not play a role in the phosphorylation‐dephosphorylation cycle.
Mutational studies of conserved residues in the dimer interface of nerve growth factor
Authors: Guo Meihua , Meyer Sheryl L. , Kaur Hardeep , Gao Jing‐Jin , Neet Kenneth E. 
Published: July 2010

AbstractAn understanding of the structure‐function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or β‐strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly fully active with 61, 120, 91, and 73% of wild‐type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20‐ and 70‐fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5‐ to 10‐fold effect on TrkA binding and no effect on low‐affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40‐fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.
Ca2+‐Binding domain VI of rat calpain is a homodimer in solution: Hydrodynamic, crystallization and preliminary X‐ray diffraction studies
Authors: Blanchard Helen , Li Yunge , Cygler Miroslaw , Kay Cyril M. , Arthur J. Simon C. , Davies Peter L. , Elce John S. 
Published: July 2010

AbstractAbstract: The 21‐kDa calcium‐binding domain (VI) of the small subunit of rat calpain II has been expressed in Escherichia coli, purified, and crystallized. Two orthorhombic crystal forms have been obtained: space group P212121 with a = 50.3, b = 56.5, c = 141.3 Å; and space group C2221 with a = 69.4, b = 73.9, c = 157.4 Å. Diffraction data have been collected to 2.4 Å. Sedimentation equilibrium, dynamic light scattering, and gel‐permeation chromatography indicate that domain VI exists as a homodimer in solution. In accordance with the protein's behavior in solution, each crystal form contains two molecules per asymmetric unit. Screening for heavy‐atom derivatives is in progress. To decrease the sensitivity to mercurials and to aid in the search for useful derivatives, Cys‐to‐Ser mutants have been prepared, expressed, and crystallized.