bluuebunny/crossref_metadata_2025 huggingface dataset:
Is human thioredoxin monomeric or dimeric?
Authors: Gronenborn Angela M. , Clore1 G. Marius , Louis John M. , Wingfield Paul T.
Published: October 2010

We have examined the molecular weight and rotational correlation time of human thioredoxin by analytical ultracentrifugation and NMR spectroscopy, respectively. Two variants of human thioredoxin were studied, namely human thioredoxin identical in amino acid sequence to the one whose NMR structure we previously determined (C62A, C69A, C73A, M74T) and human thioredoxin (C62A, C69A, C73A, M74) containing the wild‐type amino acid methionine at position 74. In both cases, the experimental data indicate that the predominant species is monomeric and we find no evidence for the existence of a well‐defined dimeric form as was observed in the recently reported crystal structure (Weichsel et al., 1996) of human thioredoxin and the C73S mutant.
Variants of ribonuclease inhibitor that resist oxidation
Authors: Kim Byung‐Moon , Schultz L.Wayne , Raines Ronald T.
Published: October 2010

Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic‐type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight‐membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10‐to 15‐fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328–Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild‐type hRI for inhibiting pancreatic‐type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.
Expression and characterization of the intact N‐terminal domain of streptokinase
Authors: Azuaga Ana I. , Woodruff Nicholas D. , Conejero‐Lara Francisco , Cox Vivienne F. , Smith Richard A.G. , Dobson Christopher M.
Published: October 2010

Proteolytic studies have enabled two of the three putative domains of the fibrinolytic protein streptokinase to be isolated and characterized (Conejero‐Lara F et al., 1996, Protein Sci 5:2583–2591). The N‐terminal domain, however, could not be isolated in these experiments because of its susceptibility to proteolytic cleavage. To complete the biophysical characterization of the domain structure of streptokinase we have overexpressed, purified, and characterized the N‐terminal region of the protein, residues 1–146. The results show this is cooperatively folded with secondary structure content and overall stability closely similar to those of the equivalent region in the intact protein.
Structure and dynamics in solution of the complex of lactobacillus casei dihydrofolate reductase with the new lipophilic antifolate drug trimetrexate
Authors: Polshakov Vladimir I. , Birdsall Berry , Frenkiel Thomas A. , Gargaro Angelo R. , Feeney James
Published: October 2010

We have determined the three‐dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate. Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N‐labeled protein. Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints. Several intermolecular protein‐ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand. At low temperature (5 °C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks. At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower‐temperature spectrum to be identified as NOEs involving ligand protons. The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conformational differences were detected between the different complexes. The 2,4‐diaminopyrimidine‐containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side‐chain orientations and local conformations of which are not greatly changed in the different complexes (similar x1 and x2 values).
Solution assembly of the pseudo‐high affinity and intermediate affinity interleukin‐2 receptor complexes
Authors: Wu Zining , Goldstein Byron , Laue Thomas M. , Liparoto Stefano F. , Nemeth Michael J. , Ciardelli Thomas L.
Published: October 2010

The high affinity interleukin‐2 receptor is composed of three cell surface subunits, IL‐2Rα, IL‐2Rβ, and IL‐2Rγ. Functional forms of the IL‐2 receptor exist, however, that enlist only two of the three subunits. On activated T‐cells, the α‐ and β‐subunits combine as a preformed heterodimer (the pseudo‐high affinity receptor) that serves to capture IL‐2. On a subpopulation of natural killer cells, the β‐ and γ‐subunits interact in a ligand‐dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled‐coil molecular recognition for the solution assembly of a heteromeric IL‐2 receptor complex. In that study, although the receptor was functional, the coiled‐coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled‐coils to generate soluble forms of both the pseudo‐high affinity and the intermediate affinity heterodimeric IL‐2 receptors. The properties of these complexes were examined and their relevance to the physiological IL‐2 receptor mechanism is discussed.
Crystal structure of thymidylate synthase A from Bacillus subtilis
Authors: Fox Kristin M. , Maley Frank , Garibian Araik , Changchien Li‐Ming , Roey Patrick Van
Published: October 2010

Thymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10‐methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents. The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli. Bacillus subtilis contains two genes for TSs, ThyA and ThyB. The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity. In ThyA TS, the active site histidine is replaced by valine. In addition, the B. subtilis enzyme has a 2.4‐fold greater kcat than the E. coli enzyme. The structure of B. subtilis thymidylate synthase in a ternary complex with 5‐fluoro‐dUMP and 5,10‐methylenetetrahydrofolate has been determined to 2.5 Å resolution. Overall, the structure of B. subtilis TS (ThyA) is similar to that of the E. coli enzyme. However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site. The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in kcat found between the two enzymes.
Two well‐defined motifs in the cAMP‐dependent protein kinase inhibitor (PKIα) correlate with inhibitory and nuclear export function
Authors: Hauer Jennifer A. , Barthe Philippe , Taylor Susan S. , Parello Joseph , Padilla André
Published: October 2010

The heat stable inhibitor of cAMP‐dependent protein kinase (PKIα) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two‐dimensional NMR spectroscopy was performed on uniformly [15N]‐labeled PKIα to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CαH chemical shifts revealed two regions of upfield chemical shifts characteristic of α‐helices. When PKIα was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIα (1–25), contains an α‐helix from residues 1–13. This helix extends by one turn the helix observed in the crystal structure of a PKIα (5–24) peptide bound to the catalytic subunit. The second region of well‐defined secondary structure, residues 35–47, overlaps with the nuclear export signal in the PKIα (26–75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well‐defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.
Energetics of solvent and ligand‐induced conformational changes in α‐lactalbumin
Authors: Griko Yuri V. , Remeta David P.
Published: October 2010

The energetics of structural changes in the holo and apo forms of α‐lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge‐charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo‐α‐lactalbumin is stable below 14 °C and undergoes a conformational change to a native‐like molten globule intermediate at temperatures above 25 °C. The denaturation of either holo‐ or apo‐α‐lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+‐binding to apo‐LA at pH 7.5 is –7.1 kJ mol–1 at 5.0 °C, which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of α‐lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo‐LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal‐binding site. Characterization of the conformational stability and ligand binding energetics of α‐lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.
Analysis of interactive packing of secondary structural elements in α/β units in proteins
Authors: Reddy Boojala V.B. , Nagarajaram Hampapathalu A. , Blundell Tom L.
Published: October 2010

An α‐helix and a β‐strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element.An analysis of all such 5,975 nonidentical α/β units in protein structures, defined at 2.5Å resolution, shows that the interaxial distance between the α‐helix and theβ‐strand is linearly correlated with the residue‐dependent function, log[(V/nda)/n‐int], where V is the volume of amino acid residues in the packing interface,nda is the normalized difference in solventaccessible contact area of the residues in packed and unpacked secondary structural elements, and n‐int is the number of residues in the packing interface. The β‐sheet unit (βu), defined as a pair of adjacent parallel or antiparallel hydrogen‐bonded β‐strands, packing with an α‐helix shows a better correlation between the interaxial distance and log (V/nda) for the residues in the packing interface.This packing relationship is shown to be useful in the prediction of interaxial distances inα/β units using the interacting residue information of equivalentα/β units of homologous proteins.It is, therefore, of value in comparative modeling of protein structures.
Functional insights from structural predictions: Analysis of the Escherichia coli genome
Authors: Rychlewski Leszek , Zhang Baohong , Godzik Adam
Published: October 2010

Fold assignments for proteins from the Escherichia coli genome are carried out using BASIC, a profile–profile alignment algorithm, recently tested on fold recognition benchmarks and on the Mycoplasma genitalium genome and PSI BLAST, the newest generation of the de facto standard in homology search algorithms. The fold assignments are followed by automated modeling and the resulting three‐dimensional models are analyzed for possible function prediction.

Close to 30% of the proteins encoded in the E. coli genome can be recognized as homologous to a protein family with known structure. Most of these homologies (23% of the entire genome) can be recognized both by PSI BLAST and BASIC algorithms, but the latter recognizes an additional 260 homologies. Previous estimates suggested that only 10–15% of E. coli proteins can be characterized this way. This dramatic increase in the number of recognized homologies between E. coli proteins and structurally characterized protein families is partly due to the rapid increase of the database of known protein structures, but mostly it is due to the significant improvement in prediction algorithms.

Knowing protein structure adds a new dimension to our understanding of its function and the predictions presented here can be used to predict function for uncharacterized proteins. Several examples, analyzed in more detail in this paper, include the DPS protein protecting DNA from oxidative damage (predicted to be homologous to ferritin with iron ion acting as a reducing agent) and the ahpC/tsa family of proteins, which provides resistance to various oxidating agents (predicted to be homologous to glutathione peroxidase).
Trifluoroethanol‐induced conformational transitions of proteins: Insights gained from the differences between α‐lactalbumin and ribonuclease A
Authors: Gast Klaus , Zirwer Dietrich , Müller‐Frohne Marlies , Damaschun Gregor
Published: October 2010

The trifluoroethanol (TFE)‐induced structural changes of two proteins widely used in folding experiments, bovine α‐lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far‐and near‐UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: α‐lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so‐called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE‐induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.
Combinatorial codons: A computer program to approximate amino acid probabilities with biased nucleotide usage
Authors: Wolf Ethan , Kim Peter S.
Published: October 2010

Using techniques from optimization theory, we have developed a computer program that approximates a desired probability distribution for amino acids by imposing a probability distribution on the four nucleotides in each of the three codon positions. These base probabilities allow for the generation of biased codons for use in mutational studies and in the design of biologically encoded libraries. The dependencies between codons in the genetic code often makes the exact generation of the desired probability distribution for amino acids impossible. Compromises are often necessary. The program, therefore, not only solves for the “optimal” approximation to the desired distribution (where the definition of “optimal” is influenced by several types of parameters entered by the user), but also solves for a number of “sub‐optimal” solutions that are classified into families ofsimilar solutions. A representative of each family is presented to the program user, who can then choose the type of approximation that is best for the intended application. The Combinatorial Codons program is available for use over the web from http://www.wi.mit.edu/kim/computing.html.
Dissection of the structural and functional role of a conserved hydration site in RNase T1
Authors: Langhorst Ulrike , Loris Remy , Denisov Vladimir P. , Doumen Jan , Roose Patrice , Maes Dominique , Halle Bertil , Steyaert Jan
Published: October 2010

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X‐ray crystallography, and 17O and 2H nuclear magnetic relaxation dispersion (NMRD). The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93Ne2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side‐chain atom, at the center of this intricate hydrogen bond network. Comparison of the 17O NMRD profiles from wild‐type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27°C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.
Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions
Authors: Breslow Esther , Mombouyran Valerie , Deeb Ruba , Zheng Changsheng , Rose John P. , Wang Bi‐Cheng , Haschemeyer Rudy H.
Published: October 2010

The structural origins of the specificity of the neurophysin hormone‐binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide α‐amino group, two factors are considered central. (1) The bound Leu side chain can establish only one‐third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.
WW: An isolated three‐stranded antiparallel β‐sheet domain that unfolds and refolds reversibly; evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state
Authors: Koepf Edward K. , Petrassi1 H. Michael , Sudol Marius , Kelly Jeffery W.
Published: October 2010

The objective of this study was to evaluate the suitability of the WW domain as a desirable model system to understand the folding and stability of an isolated three‐stranded antiparallel β‐sheet structure. The WW domain was subjected to thermal and chaotropic denaturation/reconstitution utilizing a variety of biophysical methods. This three‐stranded sheet folds reversibly and cooperatively utilizing both urea and GdnHCl as denaturants; however, the denatured state retains structure in the form of a hydrophobic cluster involving at least one aromatic side chain. In contrast to chaotropic denaturation, thermal denaturation appears to be more complete and may be a two state process. The suitability of the WW domain for future studies aimed at understanding the kinetics and thermodynamics of antiparallel β‐sheet folding clearly emerges from this initial study. The most exciting and significant result in this manuscript is the finding that the chaotropic denatured state of WW has a hydrophobic cluster as discerned by near‐UV CD evidence. The role that the denatured state plays in the folding and stability of a three‐stranded β‐sheets, and its capacity for preventing aggregation may be particularly important and is the subject of ongoing studies.
Specificity of coenzyme analogues and fragments in promoting or impeding the refolding of clostridial glutamate dehydrogenase
Authors: Aghajanian Suren , Walsh Thomas P. , Engel Paul C.
Published: October 2010

NAD+facilitates high‐yield reactivation of clostridial glutamate dehydrogenase (GDH) after unfolding in urea. The specificity of this effect has been explored by using analogues and fragments of NAD+.The adenine portion, unlike the nicotinamide portion, is important for reactivation. Alteration in the nicotinamide portion, in acetylpyridine adenine dinucleotide, has little effect, whereas loss of the 6‐NH2substitution on the adenine ring, in 6‐deamino NAD, diminishes the effectiveness of the nucleotide in promoting refolding. Also ADP‐ribose, lacking nicotinamide, promotes reactivation whereas NMN‐phosphoribose, lacking the adenine, does not.

Of the smaller fragments, those containing an adenosine moiety, and especially those with one or more phosphate groups, impede the refolding ability of NAD+, and are able to bind to the folding intermediate though unable to facilitate refolding.

These results are interpreted in terms of the known 3D structure for clostridial glutamate dehydrogenase. It is assumed that the refolding intermediate has a more or less fully formed NAD+‐binding domain but a partially disordered substrate‐binding domain and linking region. Binding of NAD+or ADP‐ribose appears to impose new structural constraints that result in completion of the correct folding of the second domain, allowing association of enzyme molecules to form the native hexamer.
The loss in hydrophobic surface area resulting from a Leu to Val mutation at the N‐terminus of the aldehyde dehydrogenase presequence prevents import of the protein into mitochondria
Authors: Hammen Philip K. , Heard Thomas S. , Waltner Mary , Weiner Henry
Published: October 2010

An apparent conservative mutation, Leu to Val, at the second residue of the rat liver mitochondrial aldehyde dehydrogenase (ALDH) presequence resulted in a precursor protein that was not imported into mitochondria. Additional mutants were made to substitute various amino acids with nonpolar side chains for Leu2. The Ile, Phe, and Trp mutants were imported to an extent similar to that of the native precursor, but the Ala mutant was imported only about one‐fourth as well. It was shown that the N‐terminal methionine was removed from the L2V mutant in a reaction catalyzed by methionine aminopeptidase. The N‐terminal methionine of native pALDH and the other mutant presequences was blocked, presumably by acetylation. Because of the difference in co‐translational modification, the L2V mutant sustained a significant loss in the available hydrophobic surface of the presequence. Import competence was restored to the L2V mutant when it was translated using a system that did not remove Met1. The removal of anArg‐Gly‐Pro helix linker segment (residues 11–14) from the L2V mutant, which shifted three leucine residues toward the N‐terminus, also restored import competence. These results lead to the conclusion that a minimum amount of hydrophobic surface area near the N‐termini of mitochondrial presequences is an essential property to determine their ability to be imported. As a result, both electrostatic and hydrophobic components must be considered when trying to understand the interactions between precursor proteins and proteins of the mitochondrial import apparatus.
Protein structural topology: Automated analysis and diagrammatic representation
Authors: Westhead David R. , Slidel Timothy W.F. , Flores Tomas P.J. , Thornton Janet M.
Published: October 2010

The topology of a protein structure is a highly simplified description of its fold including only the sequence of secondary structure elements, and their relative spatial positions and approximate orientations. This information can be embodied in a two‐dimensional diagram of protein topology, called a TOPS cartoon. These cartoons are useful for the understanding of particular folds and making comparisons between folds. Here we describe a new algorithm for the production of TOPS cartoons, which is more robust than those previously available, and has a much higher success rate. This algorithm has been used to produce a database of protein topology cartoons that covers most of the data bank of known protein structures.
Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine
Authors: Marina Alberto , Alzari Pedro M. , Bravo Jerónimo , Uriarte Matxalen , Barcelona Belén , Fita Ignacio , Rubio Vicente
Published: October 2010

The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP‐phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate‐making enzymes. A molecular 16‐stranded open β‐sheet surrounded by α‐helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site‐directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.
Folding and binding cascades: Dynamic landscapes and population shifts
Authors: Kumar Sandeep , Ma Buyong , Tsai Chung‐Jung , Sinha Neeti , Nussinov Ruth
Published: October 2010

Whereas previously we have successfully utilized the folding funnels concept to rationalize binding mechanisms (Ma B, Kumar S, Tsai CJ, Nussinov R, 1999, Protein Eng 12:713–720) and to describe binding (Tsai CJ, Kumar S, Ma B, Nussinov R, 1999, Protein Sci 8:1181–1190), here we further extend the concept of folding funnels, illustrating its utility in explaining enzyme pathways, multimolecular associations, and allostery. This extension is based on the recognition that funnels are not stationary; rather, they are dynamic, depending on the physical or binding conditions (Tsai CJ, Ma B, Nussinov R, 1999, Proc Natl Acad Sci USA 96:9970–9972). Different binding states change the surrounding environment of proteins. The changed environment is in turn expressed in shifted energy landscapes, with different shapes and distributions of populations of conformers. Hence, the function of a protein and its properties are not only decided by the static folded three‐dimensional structure; they are determined by the distribution of its conformational substates, and in particular, by the redistributions of the populations under different environments. That is, protein function derives from its dynamic energy landscape, caused by changes in its surroundings.
Antibody‐detected folding: Kinetics of surface epitope formation are distinct from other folding phases
Authors: Raman C.S. , Nall Barry T. , Jemmerson Ronald
Published: October 2010

The rate of macromolecular surface formation in yeast iso‐2 cytochrome c and its site‐specific mutant, N52I iso‐2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody‐detected (ad) kinetic phase accompanying epitope formation has Kad = 0.2 s−1 and is ∼40‐fold slower than the fastest detectable event in the folding of yeast iso‐2 cytochrome c (k2f ∼ 8 s−1), but occurs prior to the absorbance‐ and fluorescence‐detected slow folding steps (K1a ∼ 0.06 s−1; k1b ∼ 0.09 s‐1). N52I iso‐2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non‐native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso‐2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.
Kinetic evidence of an on‐pathway intermediate in the folding of lysozyme
Authors: Bai Yawen
Published: October 2010

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Iα) of hen lysozyme with α‐domain folded and β‐domain unfolded is on the folding pathway under the classical definition. lα folds to the native (N) state directly (unfolded (U) ⟺ Iα ⟺ N) without having to unfold to U and then refold to N through alternative folding pathways as in Iα ⟺ U ⟺ N.
NMR analysis of cleaved Escherichia coli thioredoxin (1–73/74–108) and its P76A variant: Cis/trans peptide isomerization
Authors: Yu Wen‐Feng , Tasayco María Luisa , Tung Chang‐Shung , Wang Hsin
Published: October 2010

Inspection of high resolution three‐dimensional (3D) structures from the protein database reveals an increasing number of cis‐Xaa‐Pro and cis‐Xaa‐Yaa peptide bonds. However, we are still far from being able to predict whether these bonds will remain cis upon single‐site substitution of Pro or Yaa and/or cleavage of a peptide bond close to it in the sequence. We have chosen oxidized Escherichia coli thioredoxin (Trx), a member of the Trx superfamily with a single α/β domain and cis P76 to determine the effect of single‐site substitution and/or cleavage on this isomer. Standard two‐dimensional (2D) NMR analysis were performed on cleaved Trx (1–73074‐108) and its P76A variant. Analysis of the NOE connectivities indicates remarkable similarity between the secondary and supersecondary structure of the noncovalent complexes and Trx. Analysis of the 2D version of the HCCH‐TOCSY and HMQC‐NOESY‐HMQC and 13C‐filtered HMQC‐NOESY spectra of cleaved Trx with uniformly 13C‐labeled 175 and P76 shows surprising conservation of both cis P76 and packing of 175 against W31. A similar NMR analysis of its P76A variant provides no evidence for cis A76 and shows only subtle local changes in both the packing of 175 and the interstrand connectivities between its most protected hydrophobic strands (β2 and β4). Indeed, a molecular simulation model for the trans P76A variant of Trx shows only subtle local changes around the substitution site. In conclusion, cleavage of R73 is insufficient to provoke cis/trans isomerization of P76, but cleavage and single‐site substitution (P76A) favors the trans isomer.
The crystal structures of human α‐thrombin complexed with active site‐directed diamino benzo[b] thiophene derivatives: A binding mode for a structurally novel class of inhibitors
Authors: Chirgadze N.Y. , Sall D.J. , Briggs S.L. , Clawson D.K. , Zhang M. , Smith G.F. , Schevitz R.W.
Published: October 2010

The crystal structures of four active site‐directed thrombin inhibitors, 1–4, in a complex with human α‐thrombin have been determined and refined at up to 2.0 Å resolution using X‐ray crystallography. These compounds belong to a structurally novel family of inhibitors based on a 2,3‐disubstituted benzo[b]thiophene structure. Compared to traditional active‐site directed inhibitors, the X‐ray crystal structures of these complexes reveal a novel binding mode. Unexpectedly, the lipophilic benzo[b]thiophene nucleus of the inhibitor appears to bind in the S1 specificity pocket. At the same time, the basic amine of the C‐3 side chain of the inhibitor interacts with the mostly hydrophobic proximal, S2, and distal, S3, binding sites. The second, basic amine side chain at C‐2 was found to point away from the active site, occupying a location between the S1 and S'1 sites. Together, the aromatic rings of the C‐2 and C‐3 side chains sandwich the indole ring of Trp60D contained in the thrombin S2 insertion loop defined by the sequence “Tyr‐Pro‐Pro‐Trp.” [The thrombin residue numbering used in this study is equivalent to that reported for chymotrypsinogen (Hartley BS, Shotton DM, 1971, The enzymes, vol. 3. New York: Academic Press. pp 323–373).] In contrast to the binding mode of more classical thrombin inhibitors (D‐Phe‐Pro‐Arg‐H, NAPAP, Argatroban), this novel class of benzo[b]thiophene derivatives does not engage in hydrogen bond formation with Gly216 of the thrombin active site. A detailed analysis of the three‐dimensional structures not only provides a clearer understanding of the interaction of these agents with thrombin, but forms a foundation for rational structure‐based drug design. The use of the data from this study has led to the design of derivatives that are up to 2,900‐fold more potent than the screening hit 1.
Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase
Authors: Knapp James E. , Carroll Donald , Lawson Janet E. , Ernst Stephen R. , Reed Lester J. , Hackert Marvin L.
Published: October 2010

The dihydrolipoamide succinyltransferase (E2o) component of the α‐ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S‐succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24‐mer, but is found as a trimer when E2o is expressed with a C‐terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 Å resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R‐factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 Å resolution. The core of tE2oCD (residues 187–396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 Å for all main‐chain atoms. The C‐terminal end of tE2oCD (residues 397–404) rotates by an average of 378 compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active‐site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein‐protein contacts that occur between the catalytic and lipoyl domains of E2o.
The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase
Authors: Sakash Jessica B. , Tsen Anita , Kantrowitz Evan R.
Published: October 2010

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site‐specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5′‐triphosphate (PTP), 6‐chloropurine 5′‐triphosphate (Cl‐PTP), 6‐mercaptopurine 5′‐triphosphate (SH‐PTP), 6‐methylpurine 5′‐triphosphate (Me‐PTP), and 1‐methyladenosine 5′‐triphosphate (Me‐ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild‐type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl‐PTP, and SH‐PTP each activate the enzyme at subsaturating concentrations of L‐aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6‐amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me‐PTP and Me‐ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2′ and 3′ ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2′‐dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2‐carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.
Deleterious effects of β‐branched residues in the S1specificity pocket ofStreptomyces griseusproteinase B (SGPB): Crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB
Authors: Bateman Katherine S. , James Michael N.G. , Anderson Stephen , Lu Wuyuan , Qasim M.A. , Laskowski Michael
Published: October 2010

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1variants in complex with the bacterial serine proteinase,Streptomyces griseusproteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three β‐branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 Å resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 Å resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The β‐branched side chains are not complementary to the shape of the S1binding pocket in SGPB, in contrast to that of the wild‐type γ‐branched P1residue for OMTKY3, Leu18I.X1angles of approximately 40° are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1pocket. Dihedral angles of +60°, ‐60°, or 180° are more commonly observed but 40° is not unfavorable for the β‐branched side chains. Thr18I Oγ1also forms a hydrogen bond with Ser195 Oγin this orientation. The Ser18I side chain adopts two alternate conformations within the S1pocket of SGPB, suggesting that the side chain is not stable in either conformation.
Dynamics of the Hck‐SH3 domain: Comparison of experiment with multiple molecular dynamics simulations
Authors: Horita David A. , Byrd R. Andrew , Zhang Weixing , Smithgall Thomas E. , Gmeiner William H.
Published: October 2010

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X‐ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481–493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 15N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S 2) analysis for regions of the protein that have experimentally well‐defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root‐mean‐square deviations among NMR‐determined structural family members or high B‐factors/low electron density in X‐ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5–10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.
A novel method of affinity‐purifying proteins using a bis‐arsenical fluorescein
Authors: Thorn Kurt S. , Naber Nartman , Matuska Marija , Cooke Roger , Vale Ronald D.
Published: October 2010

Genetically‐encoded affinity tags constitute an important strategy for purifying proteins. Here, we have designed a novel affinity matrix based on the bis‐arsenical fluorescein dye FlAsH, which specifically recognizes short α‐helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269–212). We find that kinesin tagged with this cysteine‐containing helix binds specifically to FlAsH resin and can be eluted in a fully active form. This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use. The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin. Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol. Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes.
Protein global fold determination using site‐directed spin and isotope labeling
Authors: Gaponenko Vadim , Howarth Jack W. , Gasmi‐Seabrook Genevieve , Yuan Jie , Columbus Linda , Hubbell Wayne L. , Rosevear Paul R.
Published: October 2010

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site‐directed spin labeling (SDSL) in combination with isotope enrichment to determine long‐range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well‐characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 Å were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long‐range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 å from the crystal structure. this approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.