bluuebunny/crossref_metadata_2025 huggingface dataset:
Facile transition between 310‐ and α‐helix: Structures of 8‐, 9‐, and 10‐residue peptides containing the ‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐fragment
Authors: Karle Isabella L. , Flippen‐Anderson Judith L. , Gurunath R. , Balaram P.
Published: February 2009

A structural transition from a 310‐helix to an α‐helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc‐Aib‐Trp‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐OMe, nonapeptide (B) Boc‐Trp‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐OMe, and octapeptide (C) Boc‐(Leu‐Aib‐Ala)2‐Phe‐Aib‐OMe, are completely helical in their respective crystals. At 0.9 Å resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 310‐helices with average torsional angles ϕ(N‐Cα) and Ψ(Cα–C') of –57°, –26° for C and –60°, –27° for B. The 10‐residue peptide (A) begins as a 310‐helix and abruptly changes to an α‐helix at carbonyl O(3), which is the acceptor for both a 4 → 1 hydrogen bond with N(6)H and a 5 → 1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average ϕ, Ψ angles in the decapeptide are –58°, –28° for residues 1–3 and –63°, –41 ° for residues 4–10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 310‐ to α‐helix transition with the amide I band appearing at 1,656–1,657 cm−1 in the 9‐ and 10‐residue peptides, whereas in shorter sequences the band is observed at 1,667 cm−1.
Optimization of the electrostatic interactions in proteins of different functional and folding type
Authors: Spassov Velin Z. , Karshikoff Andrej D. , Ladenstein Rudolf
Published: February 2009

The 3‐dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge‐charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge‐charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α‐helical or β‐strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge‐charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.
Pseudodihedrals: Simplified protein backbone representation with knowledge‐based energy
Authors: Dewitte R. S. , Shakhnovich E. I.
Published: February 2009

Pairwise contact energies do not explicitly take protein secondary structure into account, and so provide an incomplete description of conformational energy. In order to construct a Hamiltonian that specifically relates to protein backbone conformations, a simplified backbone angle is used. The pseudodihedral angle (the torsion angle between planes defined by 4 consecutive α‐carbon atoms) provides a simplified backbone representation and continues to manifest information about secondary‐structure elements: the pseudo‐Ramachandran plot contains helical and sheetlike regions. The distribution of pseudodihedral angles is highly sensitive to the identity of the central pair of amino acids. Therefore, a sequence‐dependent, knowledge‐based potential energy was found according to a quasichemical approximation. These functions form complementary additions to the contact potentials currently in use. This pseudodihedral potential greatly enhances the ability to design sequences that are specific to a given conformation and also improves the ability to discriminate a native conformation from many other conformations.
The three‐dimensional structures of mutants of porphobilinogen deaminase: Toward an understanding of the structural basis of acute intermittent porphyria
Authors: Brownlie Paul D. , Cooper Jon B. , Wood Steve P. , Lambert Richard , Blundell Tom L. , Louie Gordon V. , Jordan Peter M. , Warren Martin J.
Published: February 2009

Mutations in the human gene for the enzyme porphobilinogen deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3‐dimensionai structure of human porphobilinogen deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.
Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: Structure of the empty capsid assembly intermediate at 2.9 Å resolution
Authors: Basavappa R. , Filman D.J. , Syed R. , Flore O. , Icenogle J.P. , Hogle J.M.
Published: February 2009

The crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 Å resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VPO to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N‐terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N‐terminal extension of VP1, as well as portions corresponding to VP4 and the N‐terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP1 scissile bond is located some 20 Å away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoilshaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA‐dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N‐terminal network and in generating stable virions.
Probing the catalytic roles of n2‐site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis
Authors: Witmer Mark R. , Villafranca Joseph J. , Palmieri‐Young Donna
Published: February 2009

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn2+‐KD values, UV‐vis, and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn2+‐activated enzymes showed reduced kcar/Km values for ATP (>7‐fold) and for glutamate (> 10‐fold). Of the unadenylylated Mg2+‐activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20‐fold better catalyst than WTGS. We propose the n2‐site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate γ‐glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition‐state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady‐state kinetic data suggest that modifying the charge density increases the transition‐state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X‐ray structures of GS.
Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases
Authors: Saxena Ashima , Doctor Bhupendra P. , Qian Naifeng , Kovach Ildiko M. , Kozikowski A.P. , Pang Y.P. , Vellom Daniel C. , Radic Zoran , Quinn Daniel , Taylor Palmer
Published: February 2009

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site‐specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (—)‐Huperzine A was 35‐fold more potent than (+)‐Huperzine A, with K1 values of 6.2 nM and 210 nM, respectively. In addition, (—)‐Huperzine A was 88‐fold more potent in inhibiting Torpedo acetylcholinesterase than (+)‐Huperzine A, with K1 values of 0.25 μM and 22 μM, respectively. Far larger K1 values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337(330) Phe and Tyr 337(330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3‐fold increase in K1 value for the binding of Huperzine A. Molecular mechanics energy minimization of the complexes formed between each of the 2 stereoisomers of Huperzine A and fetal bovine serum acetylcholinesterase, Torpedo acetylcholinesterase, or human butyrylcholinesterase also revealed that (—)‐Huperzine A gave a better fit than (+)‐Huperzine A and implicated Tyr 337(330) in the stereoselectivity of Huperzine A.
Solution structure of the DNA‐binding domain of the heat shock transcription factor determined by multidimensional heteronuclear magnetic resonance spectroscopy
Authors: Damberger Fred F. , Pelton Jeffrey G. , Harrison Celia J. , Nelson Hillary C.M. , Wemmer David E.
Published: February 2009

The solution structure of the 92‐residue DNA‐binding domain of the heat shock transcription factor from Kluyveromyces lactis has been determined using multidimensional NMR methods. Three‐dimensional (3D) triple resonance, 1H‐13C‐13C‐1H total correlation spectroscopy, and 15N‐separated total correlation spectroscopy‐heteronuclear multiple quantum correlation experiments were used along with various 2D spectra to make nearly complete assignments for the backbone and side‐chain 1H, 15N, and 13C resonances. Five‐hundred eighty‐three NOE constraints identified in 3D 13C‐ and l5N‐separated NOE spectroscopy (NOESY)‐heteronuclear multiple quantum correlation spectra and a 4‐dimensional 13C/13C‐edited NOESY spectrum, along with 35 ø, 9 χ1, and 30 hydrogen bond constraints, were used to calculate 30 structures by a hybrid distance geometry/simulated annealing protocol, of which 24 were used for structural comparison. The calculations revealed that a 3‐helix bundle packs against a small 4‐stranded antiparallel β‐sheet. The backbone RMS deviation (RMSD) for the family of structures was 1.03 ± 0.19 Å with respect to the average structure. The topology is analogous to that of the C‐terminal domain of the catabolite gene activator protein and appears to be in the helix‐turn‐helix family of DNA‐binding proteins. The overall fold determined by the NMR data is consistent with recent crystallographic work on this domain (Harrison CJ, Bohm AA, Nelson HCM, 1994, Science 263:224) as evidenced by RMSD between backbone atoms in the NMR and X‐ray structures of 1.77 ± 0.20 Å. Several differences were identified some of which may be due to protein‐protein interactions in the crystal.
Discovering structural correlations in α‐helices
Authors: Klingler Tod M. , Brutlag Douglas L.
Published: February 2009

We have developed a new representation for structural and functional motifs in protein sequences based on correlations between pairs of amino acids and applied it to α‐helical and β‐sheet sequences. Existing probabilistic methods for representing and analyzing protein sequences have traditionally assumed conditional independence of evidence. In other words, amino acids are assumed to have no effect on each other. However, analyses of protein structures have repeatedly demonstrated the importance of interactions between amino acids in conferring both structure and function. Using Bayesian networks, we are able to model the relationships between amino acids at distinct positions in a protein sequence in addition to the amino acid distributions at each position. We have also developed an automated program for discovering sequence correlations using standard statistical tests and validation techniques. In this paper, we test this program on sequences from secondary structure motifs, namely α‐helices and β‐sheets. In each case, the correlations our program discovers correspond well with known physical and chemical interactions between amino acids in structures. Furthermore, we show that, using different chemical alphabets for the amino acids, we discover structural relationships based on the same chemical principle used in constructing the alphabet. This new representation of 3‐dimensional features in protein motifs, such as those arising from structural or functional constraints on the sequence, can be used to improve sequence analysis tools including pattern analysis and database search.
Nerve growth factor: Structure/function relationships
Authors: Bradshaw Ralph A. , Murray‐Rust Judith , Blundell Tom L. , Mcdonald Neil Q. , Lapatto Risto , Ibáñez Carlos F.
Published: February 2009

Nerve growth factor (NGF), which has a tertiary structure based on a cluster of 3 cystine disulfides and 2 very extended, but distorted β‐hairpins, is the prototype of a larger family of neurotrophins. Prior to the availability of cloning techniques, the mouse submandibular gland was the richest source of NGF and provided sufficient material to enable its biochemical characterization. It binds as a dimer to at least 2 cell‐surface receptor types expressed in a variety of neuronal and non‐neuronal cells. Residues involved in these interactions and in the maintenance of tertiary and quaternary structure have been identified by chemical modification and site‐directed mutagenesis, and this information can be related to their location in the 3‐dimensional structure. For example, interactions between aromatic residues contribute to the stability of the NGF dimer, and specific surface lysine residues participate in receptor contacts. The conclusion from these studies is that receptor interactions involve broad surface regions, which may be composed of residues from both protomers in the dimer.
Common structural features of the luxF protein and the subunits of bacterial luciferase: Evidence for a (βα)8 fold in luciferase
Authors: Moore Stanley A. , James Michael N. G.
Published: February 2009

The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique β/α barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (βα)8 barrel fold with at least 2 excursions from the (βα)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct “pockets” of NFP, which are located at hydrophobic interfaces between the β‐strands and α‐helices. Several tight turns joining the C‐termini of β‐strands and the N‐termini of α‐helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N‐cap residues are the most strongly conserved structural features. A partial model of the luciferase β subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 Å resolution.
Fast folding of a prototypic polypeptide: The immunoglobulin binding domain of streptococcal protein G
Authors: Kuszewski John , Clore G. Marius , Gronenborn Angela M.
Published: February 2009

The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GB1) of streptococcal protein G has been investigated by quenched‐flow deuterium‐hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND‐NH exchange up to 10‐fold higher than that expected for a random coil, occurs within the dead time (≤1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half‐life of 5.2 ms at 5 °C.
Surfactant effects on protein structure examined by electrospray ionization mass spectrometry
Authors: Loo R. R. Ogorzalek , Dales N. , Andrews P. C.
Published: February 2009

Electrospray ionization mass spectrometry (ESI‐MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI‐MS and on protein structure under ESI‐MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H2O and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n‐dodecyl‐β‐D‐gluco‐pyranoside, at 0.1% to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI‐MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI‐MS. However, several of the surfactants caused significant shifts in the charge‐state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI‐MS, can be useful for protein structure studies, if care is used in the interpretation of the results.
Glu‐50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure
Authors: Tauc Patrick , Keiser Richard T. , Kantrowitz Evan R. , Vachette Patrice
Published: February 2009

Glu‐50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high‐activity high‐affinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu‐50 (Glu‐50 → Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444–1451). A study of the structural consequences of replacing Glu‐50 by alanine using solution X‐ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so‐called T quaternary conformation as is the wild‐type enzyme. In the presence of a saturating concentration of the bisubstrate analog N‐phosphonacetyl‐L‐aspartate (PALA), the mutant enzyme is in the same, so‐called R quaternary conformation as the wild‐type enzyme. However, the Glu‐50 → Ala enzyme differs from the wild‐type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steady‐state kinetic studies indicate that, in contrast to the wild‐type enzyme, the Glu‐50 → Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu‐50 are critical for the creation of the high‐activity, high‐affinity R state of the enzyme.
Analysis of hydrophobicity in the α and β chemokine families and its relevance to dimerization
Authors: Covell David G. , Smythers Gary W. , Gronenborn Angela M. , Clore G. Marius
Published: February 2009

The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, α and β, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25–70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 α and 9 β chemokine sequences, based on the coordinates of the prototypic α and β chemokines, interleukin‐8 (IL‐8), and human macrophage inflammatory protein‐10 (hMIP‐10), respectively, is presented. The monomers of the α and β chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the α and β chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the α and β chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL‐8 and hMIP‐1β are preserved for all the α and β chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the α and β chemokine subfamilies.
Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein
Authors: Seefeldt Lance C.
Published: February 2009

Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum‐iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site‐directed mutagenesis. The altered FePs were expressed in A. vinelandii in place of the wild‐type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild‐type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments. In addition, it was found that changing Arg 140 and Lys 143 to Gln resulted in a significant uncoupling of MgATP hydrolysis from intercomponent electron transfer rates between FeP and MoFeP. The wild‐type complex was found to hydrolyze 2.5 MgATP per electron transferred, whereas the R140Q and K143Q proteins, when coupled to wild‐type MoFeP, required 6 and 31 MgATP for each electron transferred, respectively. It is concluded that both Arg 140 and Lys 143 of the A. vinelandii nitrogenase FeP are important in the docking interaction with the MoFeP and that these residues probably function in aligning the FeP with the MoFeP for intercomponent electron transfer. A model is discussed in which these positively charged amino acids of the FeP might interact with negatively charged amino acids (Asp and Glu) on the MoFeP near its 8Fe cluster.
The role of glycine (residue 89) in the central helix of EF‐hand protein troponin‐C exposed following amino‐terminal α‐helix deletion
Authors: Ding Xiao‐Ling , Akella Árvind Babu , Su Hong , Gulati Jagdish
Published: February 2009

Because an N‐terminal α‐helical (N‐helix) arm and a KGK‐triplet (residues 88KGK90) in the central helix of troponin‐C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure‐function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N‐helix and the KGK‐triplet in TnC. Noticeably, the amino acid compositions vary between the N‐helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK‐triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N‐helix (mutant ΔNt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90‐triplet (mutant ΔNtΔKGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685–11690). Using this functional test, we find that replacement of Gly‐89 with a Leu or an Ala could also overcome the contractile defect associated with N‐helix deletion. On the other hand, replacement of the skeletal TnC N‐helix with cardiac type N‐helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly‐89 residue in skeletal TnC and suggest that the N‐terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N‐helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms.
Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease
Authors: Xie Dong , Freire Ernesto , Fox Robert
Published: February 2009

High‐sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117 → Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2‐state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the β subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0–100 °C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure‐based thermodynamic calculations also predict that the most probable intermediate is one in which the β subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.
Cavities and packing at protein interfaces
Authors: Hubbard Simon J. , Argos Patrick
Published: February 2009

An analysis of internal packing defects or “cavities” (both empty and water‐containing) within protein structures has been undertaken and includes 3 cavity classes: within domains, between domains, and between protein subunits. We confirm several basic features common to all cavity types but also find a number of new characteristics, including those that distinguish the classes. The total cavity volume remains only a small fraction of the total protein volume and yet increases with protein size. Water‐filled “cavities” possess a more polar surface and are typically larger. Their constituent waters are necessary to satisfy the local hydrogen bonding potential. Cavity‐surrounding atoms are observed to be, on average, less flexible than their environments. Intersubunit and inter‐domain cavities are on average larger than the intradomain cavities, occupy a larger fraction of their resident surfaces, and are more frequently water‐filled. We observe increased cavity volume at domain‐domain interfaces involved with shear type domain motions. The significance of interfacial cavities upon subunit and domain shape complementarity and the protein docking problem, as well as in their structural and functional role in oligomeric proteins, will be discussed. The results concerning cavity size, polarity, solvation, general abundance, and residue type constituency should provide useful guidelines for protein modeling and design.
Hydrogen exchange in BPTI variants that do not share a common disulfide bond
Authors: Schulman Brenda A. , Kim Peter S.
Published: February 2009

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30‐51, 5‐55, and 14‐38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5‐55]A]a and [30‐51; 14‐38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide‐bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native‐like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI foldiing intermediates containing either the 5‐55 or the 30‐51 disulfide bond.
The role of transmembrane domain III in the lactose permease of escherichia coli
Authors: Sahin‐Tóth Miklós , Frillingos Stathis , Bibi Eitan , Gonzalez Alberto , Ronald Kaback H.
Published: February 2009

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys‐scanning mutagenesis. Using a functional mutant devoid of Cys residues (C‐less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty‐one of the 25 mutants accumulate lactose to >70% of the steady‐state exhibited by C‐less permease, and an additional 3 mutants transport to lower, but significant levels (40–60% of C‐less). Cys replacement for Leu 76 results in low transport activity (18% of C‐less). However, when placed in the wild‐type background, mutant Leu 76 → Cys exhibits highly significant rates of transport (55% of wild type) and steady‐state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N‐ethylmaleimide show that mutant Gly 96 → Cys is rapidly inactivated, whereas the other single‐Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96 → Cys permease is enhanced at least 2‐fold in the presence of β‐galactopyranosyl 1‐thio‐β,D‐galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.
Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin
Authors: Wills Fiona L. , Mccubbin William D. , Kay Cyril M. , Gimona Mario , Strasser Peter
Published: February 2009

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino‐terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2–51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl‐terminal truncated mutant of calponin comprising residues 1–228 (CP 1–228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1–228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1–228 in a Ca2+‐dependent fashion, indicating that there is a second site of interaction between residues 52–228. Temperature denaturation of the carboxyl‐terminal truncated fragment compared with whole calponin show that the carboxyl‐terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45–228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.
Dissecting the energetics of an antibody‐antigen interface by alanine shaving and molecular grafting
Authors: Jin Lei , Wells James A.
Published: February 2009

Alanine‐scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) for binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells JA, 1992, J Mol Biol 226:851–865). To further analyze the energetic importance of residues surrounding these five, we mutated all neighboring residues to alanine in groups of 7–16 (a procedure we call alanine shaving). Even the most extremely mutated variant, with 16 alanine substitutions, caused less than a 10‐fold reduction in binding affinity to MAb3. By comparison, mutating any 1 of the 5 primary determinants to alanine caused a 6‐ to >500‐fold reduction in affinity. Replacing any of the 4 charged residues (Arg 8, Arg 16, Asp 112, and Asp 116) with a homologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as large a reduction in affinity as the corresponding alanine replacement. It was possible to graft the 5 primary binding determinants onto a nonbinding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10‐fold less tightly than hGH to MAb3 but bound as well as hGH when 2 additional framework mutations were introduced. Attempts to recover binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4‐helix bundle motif, such as human prolactin (23% sequence identity) or granulocyte colony‐stimulating factor, were unsuccessful. These studies show that only a small number of side chains on hGH confers tight binding affinity to MAb3 and that substituting or transplanting them onto another scaffold has narrow design tolerances.
Determinants of protein side‐chain packing
Authors: Tanimura Ryuji , Kidera Akinori , Nakamura Haruki
Published: February 2009

The problem of protein side‐chain packing for a given backbone trace is investigated using 3 different prediction models. The first requires an exhaustive search of all possible combinations of side‐chain conformers, using the dead‐end elimination theorem. The second considers only side‐chain‐backbone interactions, whereas the third neglects side‐chain‐backbone interactions and instead keeps side‐chain‐side‐chain interactions. Predictions of side‐chain conformations for 11 proteins using all 3 models show that removal of side‐chain‐side‐chain interactions does not cause a large decrease in the prediction accuracy, whereas the model having only side‐chain‐side‐chain interactions still retains a significant level of accuracy. These results suggest that the 2 classes of interactions, side‐chain‐backbone and side‐chain‐side‐chain, are consistent with each other and work concurrently to stabilize the native conformations. This is confirmed by analyses of energy spectra of the side‐chain conformations derived from the fourth prediction model, the Independent model, which gives almost the same quality of the prediction as the dead‐end elimination. The analyses indicate that the 2 classes of interactions simultaneously increase the energy difference between the native and nonnative conformations.
Molecular evolution and domain structure of plasminogen‐related growth factors (HGF/SF and HGF1/MSP)
Authors: Donate Luis Enrique , Srinivasan N. , Sowdhamini R. , Gherardi Ermanno , Blundell Tom L. , Aparicio Samuel
Published: February 2009

Plasminogen‐related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor‐like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron–exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N‐terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide‐bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix‐strand‐helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three‐dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine‐binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.
Conservation of polyproline II helices in homologous proteins: Implications for structure prediction by model building
Authors: Adzhubei Alexei A. , Sternberg Michael J.E.
Published: February 2009

Left‐handed polyproline II (PPII) helices commonly occur in globular proteins in segments of 4–8 residues. This paper analyzes the structural conservation of PPII‐helices in 3 protein families: serine proteinases, aspartic proteinases, and immunoglobulin constant domains. Calculations of the number of conserved segments based on structural alignment of homologous molecules yielded similar results for the PPII‐helices, the α‐helices, and the β‐strands. The PPII‐helices are consistently conserved at the level of 100–80% in the proteins with sequence identity above 20% and RMS deviation of structure alignments below 3.0 Å. The most structurally important PPII segments are conserved below this level of sequence identity. These results suggest that the PPII‐helices, in addition to the other 2 secondary structure classes, should be identified as part of structurally conserved regions in proteins. This is supported by similar values for the local RMS deviations of the aligned segments for the structural classes of PPII‐helices, α‐helices, and β‐strands. The PPII‐helices are shown to participate in supersecondary elements such as PPII‐helix/α‐helix. The conservation of PPII‐helices depends on the conservation of a supersecondary element as a whole. PPII‐helices also form links, possibly flexible, in the interdomain regions. The role of the PPII‐helices in model building by homology is 2‐fold: they serve as additional conserved elements in the structure allowing improvement of the accuracy of a model and provide correct chain geometry for modeling of the segments equivalenced to them in a target sequence. The improvement in model building is demonstrated in 2 test studies.
High‐Sensitivity sequencing of large proteins: Partial structure of the rapamycin‐fkbp12 target
Authors: Erdjument‐Bromage Hediye , Lui Mary , Tempst Paul , Sabatini David M. , Snyder Solomon H.
Published: February 2009

We report on studies leading to refinements of various steps of the protein internal sequencing process. Specifically, the developments comprise (1) higher‐sensitivity chemical sequencing through background reduction; (2) improved peptide recovery from rapid in situ digests of nanogram amount, nitrocellulose‐bound proteins; and (3) accurate UV spectroscopic identification of Trp‐ and Cys‐containing peptides. In addition, we describe strategies for 2‐dimensional liquid chromatographic peptide isolation from complex mixtures and a multi‐analytical approach to peptide sequence analysis (Edman sequencing, matrix‐assisted laser desorption mass spectrometry, and UV spectroscopy). Both strategies were applied in tandem to the primary structural analysis of a gel‐purified, 250‐kDa protein (mammalian target of rapamycin‐FKBP12 complex), available in low picomolar quantities only. More than 300‐amino acids worth of sequence was obtained in mostly uninterrupted stretches, several containing Trp, Cys, His, and Ser. That information has allowed the matching of a biological function of a mammalian protein to a yeast gene product with a well‐characterized mutant phenotype. The results also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.
Crystallization and preliminary x‐ray analysis of glucose‐fructose oxidoreductase from zymomonas mobilis
Authors: Loos Heidi , Ermler Ulrich , Sprenger Georg A. , Sahm Hermann
Published: February 2009

Glucose‐fructose oxidoreductase (E.C. 1.1.99.‐) from the ethanol‐producing Gram‐negative bacterium Zymomonas mobilis is a periplasmic, soluble enzyme that forms a homotet‐ramer of 160 kDa with one NADP(H) cofactor per subunit that is tightly, but noncovalently, bound. The enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. The obtained crystals belong to the space group P21212, with unit cell constants of 84.6 Å, 94.1 Å, and 117.0 Å, consistent with two monomers in the asymmetric unit. They diffract to a resolution of about 2 Å and are suitable for X‐ray structure determination.