bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*)
Authors: Rodgers Karla K. , Coleman Joseph E.
Published: July 2010

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

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

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

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

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

We present a method based on hierarchical self‐organizing maps (SOMs) for recognizing patterns in protein sequences. The method is fully automatic, does not require prealigned sequences, is insensitive to redundancy in the training set, and works surprisingly well even with small learning sets. Because it uses unsupervised neural networks, it is able to extract patterns that are not present in all of the unaligned sequences of the learning set. The identification of these patterns in sequence databases is sensitive and efficient.

The procedure comprises three main training stages. In the first stage, one SOM is trained to extract common features from the set of unaligned learning sequences. A feature is a number of ungapped sequence segments (usually 4–16 residues long) that are similar to segments in most of the sequences of the learning set according to an initial similarity matrix. In the second training stage, the recognition of each individual feature is refined by selecting an optimal weighting matrix out of a variety of existing amino acid similarity matrices. In a third stage of the SOM procedure, the position of the features in the individual sequences is learned. This allows for variants with feature repeats and feature shuffling.

The procedure has been successfully applied to a number of notoriously difficult cases with distinct recognition problems: helix‐turn‐helix motifs in DNA‐binding proteins, the CUB domain of developmentally regulated proteins, and the superfamily of ribokinases. A comparison with the established database search procedure PROFILE (and with several others) led to the conclusion that the new automatic method performs satisfactorily.
Conformation of the hypervariable region L3 without the key proline residue
Authors: Guarné Alba , Bravo Jerónimo , Calvo Javier , Lozano Francisco , Vives Jordi , Fita Ignasi
Published: July 2010

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

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

The structure of the phosphorylated form of the histidine‐containing phosphocarrier protein HPr from Escherichia coli has been solved by NMR and compared with that of unphosphorylated HPr. The structural changes that occur upon phosphorylation of His 15, monitored by changes in NOE patterns, 3JNHHα‐coupling constants, and chemical shifts, are limited to the region around the phosphorylation site. The His 15 backbone torsion angles become strained upon phosphorylation. The release of this strain during the phosphoryl‐transfer to Enzyme II facilitates the transport of carbohydrates across the membrane.

From an X‐ray study of Streptococcus faecalis HPr (Jia Z, Vandonselaar M, Quail JW, Delbaere LTJ, 1993, Nature 361:94–97), it was proposed that the observed torsion‐angle strain at residue 16 in unphosphorylated S. faecalis HPr has a role to play in the protein's phosphocarrier function. The model predicts that this strain is released upon phosphorylation. Our observations on E. coli HPr in solution, which shows strain only after phosphorylation, and the fact that all other HPrs studied thus far in their unphosphorylated forms show no strain either, led us to investigate the possibility that the crystal environment causes the strain in S. faecalis HPr. A 1‐ns molecular dynamics simulation of S. faecalis HPr, under conditions that mimic the crystal environment, confirms the observations from the X‐ray study, including the torsion‐angle strain at residue 16. The strain disappeared, however, when S. faecalis HPr was simulated in a water environment, resulting in an active site configuration virtually the same as that observed in all other unphosphorylated HPrs. This indicates that the torsion‐angle strain at Ala 16 in S. faecalis HPr is a result of crystal contacts or conditions and does not play a role in the phosphorylation‐dephosphorylation cycle.
Mutational studies of conserved residues in the dimer interface of nerve growth factor
Authors: Guo Meihua , Meyer Sheryl L. , Kaur Hardeep , Gao Jing‐Jin , Neet Kenneth E.
Published: July 2010

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

Abstract: The 21‐kDa calcium‐binding domain (VI) of the small subunit of rat calpain II has been expressed in Escherichia coli, purified, and crystallized. Two orthorhombic crystal forms have been obtained: space group P212121 with a = 50.3, b = 56.5, c = 141.3 Å; and space group C2221 with a = 69.4, b = 73.9, c = 157.4 Å. Diffraction data have been collected to 2.4 Å. Sedimentation equilibrium, dynamic light scattering, and gel‐permeation chromatography indicate that domain VI exists as a homodimer in solution. In accordance with the protein's behavior in solution, each crystal form contains two molecules per asymmetric unit. Screening for heavy‐atom derivatives is in progress. To decrease the sensitivity to mercurials and to aid in the search for useful derivatives, Cys‐to‐Ser mutants have been prepared, expressed, and crystallized.
Prediction of protein complexes using empirical free energy functions
Authors: Weng Zhiping , Vajda Sandor , Delisi Charles
Published: July 2010

A long sought goal in the physical chemistry of macromolecular structure, and one directly relevant to understanding the molecular basis of biological recognition, is predicting the geometry of bimolecular complexes from the geometries of their free monomers. Even when the monomers remain relatively unchanged by complex formation, prediction has been difficult because the free energies of alternative conformations of the complex have been difficult to evaluate quickly and accurately. This has forced the use of incomplete target functions, which typically do no better than to provide tens of possible complexes with no way of choosing between them. Here we present a general framework for empirical free energy evaluation and report calculations, based on a relatively complete and easily executable free energy function, that indicate that the structures of complexes can be predicted accurately from the structures of monomers, including close sequence homologues. The calculations also suggest that the binding free energies themselves may be predicted with reasonable accuracy. The method is compared to an alternative formulation that has also been applied recently to the same data set. Both approaches promise to open new opportunities in macromolecular design and specificity modification.
High level expression and crystallization of recombinant human cathepsin S
Authors: Brömme Dieter , Mcgrath Mary E.
Published: July 2010

We have expressed active human cathepsin S to 60 mg/L in Sf9 cells using a baculovirus system. Production of milligram quantities has facilitated crystallographic studies to determine the structure of this enzyme, which has unique properties among lysosomal cysteine proteinases. Recombinant, irreversibly inhibited cathepsin S was crystallized from ammonium phosphate at 17 °C. The crystals diffract to at least 2.3 Å, and belong to the orthorhombic crystal system with a primitive lattice. Approximate cell dimensions are: a = 37.7 Å, b = 73.9 Å, and c = 106.7 Å. There is most likely one molecule per asymmetric unit.
Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of minibody
Authors: Zappacosta Francesca , Pessi Antonello , Bianchi Elisabetta , Venturini Sara , Sollazzo Maurizio , Tramontano Anna , Marino Gennaro , Pucci Piero
Published: July 2010

A strategy that combines limited proteolysis experiments and mass spectrometrie analysis of the fragments generated has been developed to probe protease‐accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64‐residue protein consisting of a β‐sheet scaffold based on the heavy‐chain variable‐domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X‐ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single‐residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X‐ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.
Interaction of subtilisins with serpins
Authors: Komiyama Tomoko , Grøn Hanne , Salvesen Guy S. , Pemberton Philip A.
Published: July 2010

Serpins are well‐characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that α1proteinase inhibitor inhibits subtilisin Carlsberg and proteinase K, and α1antichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0–45 °C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.
Enzymatic and fluorescence studies of four single‐tryptophan mutants of rat testis fructose 6‐phosphate,2‐kinase:fructose 2,6‐bisphosphatase
Authors: Watanabe Fusao , Uyeda Kosaku , Jameson David M.
Published: July 2010

In order to determine environments around four tryptophan residues, located in the N‐terminus, in the kinase and in the phosphatase domains of rat testis Fru 6‐P,2‐kinase:Fru 2,6‐bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site‐directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild‐type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5× that of the wild‐type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild‐type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild‐type enzyme. The polarization in the presence of Fru 6‐P affected only Trp 15, which suggested that it is located near the Fru 6‐P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6‐P,2‐kinase and Fru 2,6‐bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.
Protein folding for realists: A timeless phenomenon
Authors: Shortle David , Wang Yi , Gillespie Joel R. , Wrabl James O.
Published: July 2010

Future research on protein folding must confront two serious dilemmas. (1) It may never be possible to observe at high resolution the very important structures that form in the first few milliseconds of the refolding reaction. (2) The energy functions used to predict structure from sequence will always be approximations of the true energy function. One strategy to resolve both dilemmas is to view protein folding from a different perspective, one that no longer emphasizes time and unique trajectories through conformation space. Instead, free energy replaces time as the reaction coordinate, and ensembles of equilibrium states of partially folded proteins are analyzed in place of trajectories of one protein chain through conformation space, either in vitro or in silico. Initial characterization of the folding of staphylococcal nuclease within this alternative conceptual framework has led to an equilibrium folding pathway with several surprising features. In addition to the finding of two bundles of four hydrophobic segments containing both native and non‐native interactions, a gradient in relative stability of different substructures has been identified, with the most stable interactions located toward the amino terminus and the least stable toward the carboxy terminus. Hydrophobic bundles with up‐down topology and stability gradients may be two examples of numerous tactics used by proteins to facilitate rapid folding and minimize aggregation. As NMR methods for structural analysis of partially folded proteins are refined, higher resolution descriptions of the structure and dynamics of the polypeptide chain outside the native state may provide many insights into the processes and energetics underlying the self‐assembly of folded structure.
Mobile unnatural amino acid side chains in the core of staphylococcal nuclease
Authors: Wynn Richard , Harkins Paul C. , Richards Frederic M. , Fox Robert O.
Published: July 2010

The structures of several variants of staphylococcal nuclease with long flexible unnatural amino acid side chains in the hydrophobic core have been determined by X‐ray crystallography. The unnatural amino acids are disulfide moieties between the lone cysteine residue in V23C nuclease and methane, ethane, 1‐n‐propane, 1‐n‐butane, 1‐n‐pentane, and 2‐hydroxyethyl thiols. We have examined changes in the core packing of these mutants. Side chains as large as the 1‐n‐propyl cysteine disulfide can be incorporated without perturbation of the structure. This is due, in part, to cavities present in the wild‐type protein. The longest side chains are not well defined, even though they remain buried within the protein interior. These results suggest that the enthalpy‐entropy balance that governs the rigidity of protein interiors favors tight packing only weakly. Additionally, the tight packing observed normally in protein interiors may reflect, in part, the limited numbers of rotamers available to the natural amino acids.
Crystal structure of cutinase covalently inhibited by a triglyceride analogue
Authors: Longhi Sonia , Knoops‐Mouthuy Edith , Cambillau Christian , Mannesse Maurice , Verheij Hubertus M. , De Haas Gerard H. , Egmond Maarten
Published: July 2010

Cutinase from Fusarium solani is a lipolytic enzyme that hydrolyses triglycerides efficiently. All the inhibited forms of lipolytic enzymes described so far are based on the use of small organophosphate and organophosphonate inhibitors, which bear little resemblance to a natural triglyceride substrate. In this article we describe the crystal structure of cutinase covalently inhibited by (R)‐1,2‐dibutyl‐carbamoylglycero‐3‐O‐p‐nitrophenylbutyl‐phosphonate, a triglyceride analogue mimicking the first tetrahedral intermediate along the reaction pathway. The structure, which has been solved at 2.3 Å, reveals that in both the protein molecules of the asymmetric unit the inhibitor is almost completely embedded in the active site crevice. The overall shape of the inhibitor is that of a fork: the two dibutyl‐carbamoyl chains point towards the surface of the protein, whereas the butyl chain bound to the phosphorous atom is roughly perpendicular to the sn‐1 and sn‐2 chains. The sn‐3 chain is accommodated in a rather small pocket at the bottom of the active site crevice, thus providing a structural explanation for the preference of cutinase for short acyl chain substrates.
A desolvation barrier to hydrophobic cluster formation may contribute to the rate‐limiting step in protein folding
Authors: Rank Jeffrey A. , Baker David
Published: July 2010

To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water‐excluding cluster relative to both the close‐packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent‐accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.
Identification of a novel phosphatase sequence motif
Authors: Stukey Joseph , Carman George M.
Published: July 2010

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP‐(X12‐54)‐PSGH‐(X31‐54))‐SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose‐6‐phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium‐containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vana‐date, a compound structurally similar to phosphate. Structure‐function analysis of the human glucose‐6‐phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.
An engineered amino‐terminal domain of yeast phosphoglycerate kinase with native‐like structure
Authors: Sherman Mark A. , Chen Yuan , Mas Maria T.
Published: July 2010

Previous studies have suggested that the carboxy‐terminal peptide (residues 401–415) and interdomain helix (residues 185–199) of yeast phosphoglycerate kinase, a two‐domain enzyme, play a role in the folding and stability of the amino‐terminal domain (residues 1–184). A deletion mutant has been created in which the carboxy‐terminal peptide is attached to the amino‐terminal domain (residues 1–184) plus interdomain helix (residues 185–199) through a flexible peptide linker, thus eliminating the carboxy‐terminal domain entirely. CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N‐domain is soluble, monomeric, compactly folded, native‐like in structure, and capable of binding the substrate 3‐phosphoglycerate with high affinity in a saturable manner. The midpoint of the guanidine‐induced unfolding transition was the same as that of the native two‐domain protein (Cm ∼ 0.8 M). The free energy change associated with guanidine‐induced unfolding was one‐third that of the native enzyme, in agreement with previous studies that evaluated the intrinsic stability of the N‐domain and the contribution of domain‐domain interactions to the stability of PGK. These observations suggest that the C‐terminal peptide and interdomain helix are sufficient for maintaining a native‐like fold of the N‐domain in the absence of the C‐domain.
Amino acid sequence of bovine gamma E (IVa) lens crystallin
Authors: Kilby Greg W. , Sheil Margaret M. , Shaw Denis , Harding John J. , Truscott Roger J. W.
Published: July 2010

When electrospray ionization mass spectrometry (ES‐MS) was used to analyze purified bovine gamma E (γIVa)‐crystallin, it yielded a relative molecular mass (Mr) of 20,955 ± 5. This mass is significantly different from that calculated from the published sequence (Mr 20,894) (White HE et al., 1989, J Mol Biol 207:217–235). Further, ES‐MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203–208). The entire protein sequence of γE crystallin has therefore been studied via a combination of ES‐MS, ES‐MS/MS, and Edman amino acid sequencing. The corrected sequence gives an Mr of 20,955.3, which matches that obtained by ES‐MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine γ‐crystallin by R. Hay (pers. comm.).
Production, crystallization, and preliminary X‐ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1‐47) of troponin I
Authors: Saijo Yumiko , Takeda Soichi , Scherer Anna , Kobayashi Tomoyoshi , Maéda Yuichiro , Taniguchi Hisaaki , Yao Min , Wakatsuki Soichi
Published: July 2010

Troponin is a ternary protein complex consisting of subunits TnC, TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli‐expressed rabbit skeletal muscle TnC and fragment 1‐47 of TnI, which is obtained by chemical cleavage of an E. coli‐expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P31(2)21, with unit cell dimensions a = b = 48.2 Å, c = 162 Å. The asymmetric unit presumably contains one CI47 complex. Soaking with p‐chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 Å resolution, with completeness of 91% and Rmerge = 6.4%. The crystals of PCMBS‐derivative should be suitable for X‐ray studies using the multiple‐wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.
Archaeal phosphoproteins. Identification of a hexosephosphate mutase and the α‐subunit of succinyl‐CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus
Authors: Solow Barbara , Bischoff Kenneth M. , Zylka Mark J. , Kennelly Peter J.
Published: July 2010

When soluble extracts from the extreme acidophilic archaeon Sulfolobus solfataricus were incubated with [γ‐32P]ATP, several radiolabeled polypeptides were observed following SDS‐PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35‐kDa one, whose protein‐phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+ versus Mn2+, and the choice of incubation temperature. The 35‐ and 50‐kDa phosphoproteins were purified and their amino‐terminal sequences determined. The former polypeptide's amino‐terminal sequence closely matched a conserved portion of the α‐subunit of succinyl‐CoA synthetase, which forms an acid‐labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50‐kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro‐Gly‐Thr‐Asp/Ser‐Gly‐Val/Leu‐Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6‐phosphate to donate its phosphoryl group to the protein.
X‐ray structures of three interface mutants of γB‐crystallin from bovine eye lens
Authors: Palme Stefan , Slingsby Christine , Jaenicke Rainer
Published: July 2010

γB‐crystallin consists of two domains each comprising two “Greek key” motifs. Both domains fold independently, and domain interactions contribute significantly to the stability of the C‐terminal domain. In a previous study (Palme S et al., 1996, Protein Sci 6:1529‐1636) it was shown that Phe56 from the N‐terminal domain, a residue involved in forming a hydrophobic core at the domain interface, effects the interaction of the two domains, and therefore, the stability of the C‐terminal domain. Ala or Asp at position 56 drastically decreased the stability of the C‐terminal domain, whereas Trp had a more moderate effect. In this article we present the X‐ray structures of these interface mutants and correlate them with the stability data. The mutations do not effect the overall structure of the molecule. No structural changes are observed in the vicinity of the replaced residue, suggesting that the local structure is too rigid to allow compensations for the amino acid replacements. In the mutants γB‐F56A and ‐F56D, a solvent‐filled groove accessible to the bulk solvent is created by the replacement of the bulky Phe side chain. In γB‐F56W, the pyrrole moiety of the indole ring replaces the phenyl side chain of the wild type. With the exception of γB‐F56W, there is a good correlation between the hydrophobicity of the amino acid at position 56 according to the octanol scale and the stability of the C‐terminal domain. In γB‐F56W, the C‐terminal domain is less stable than estimated from the hydrophobicity, presumably because the ring nitrogen (Nϵ1) has no partner to form hydrogen bonds. The data suggest that the packing of hydrophobic residues in the interface core is important for domain interactions and the stability of γB‐crystallin. Apparently, for protein stability, the same principles apply for hydrophobic cores within domains and at domain interfaces.
Locally accessible conformations of proteins: Multiple molecular dynamics simulations of crambin
Authors: Caves Leo S. D. , Evanseck Jeffrey D. , Karplus Martin
Published: July 2010

Multiple molecular dynamics (MD) simulations of crambin with different initial atomic velocities are used to sample conformations in the vicinity of the native structure. Individual trajectories of length up to 5 ns sample only a fraction of the conformational distribution generated by ten independent 120 ps trajectories at 300 K. The backbone atom conformational space distribution is analyzed using principal components analysis (PCA). Four different major conformational regions are found. In general, a trajectory samples only one region and few transitions between the regions are observed. Consequently, the averages of structural and dynamic properties over the ten trajectories differ significantly from those obtained from individual trajectories. The nature of the conformational sampling has important consequences for the utilization of MD simulations for a wide range of problems, such as comparisons with X‐ray or NMR data. The overall average structure is significantly closer to the X‐ray structure than any of the individual trajectory average structures. The high frequency (less than 10 ps) atomic fluctuations from the ten trajectories tend to be similar, but the lower frequency (100 ps) motions are different. To improve conformational sampling in molecular dynamics simulations of proteins, as in nucleic acids, multiple trajectories with different initial conditions should be used rather than a single long trajectory.
The methanol‐induced transition and the expanded helical conformation in hen lysozyme
Authors: Kamatari Yuji O. , Akasaka Kazuyuki , Konno Takashi , Kataoka Mikio
Published: July 2010

Methanol‐induced conformational transitions of hen egg white lysozyme were investigated with a combined use of far and near‐UV CD and NMR spectroscopies, ANS binding and small‐angle X‐ray scattering. Addition of methanol induced no global change in the native conformation itself, but induced a transition from the native state to the denatured state which was highly cooperative, as shown by the coincidence of transition curves monitored by the far‐and near‐UV CD spectroscopy, by isodichroic points in the far‐ and near‐UV CD spectra and by the concomitant disappearance of individual 1H NMR signals of the native state. The ANS binding experiments could detect no intermediate conformer similar to the molten globule state in the process of the methanol denaturation. However, at high concentration of methanol, e.g., 60% (v/v) methanol/water, a highly helical state (H) was realized. The H state had a helical content much higher than the native state, monitored by far‐UV CD spectroscopy, and had no specific tertiary structure, monitored both by near‐UV CD and NMR spectroscopy. The radius of gyration in the H state, 24.9 Å, was significantly larger than that in the native state (15.7 Å). The Kratky plot for the H state did not show a clear peak and was quite similar to that for the urea‐denatured state, indicating a complete lack of globularity. Thus we conclude that the H state has a considerably expanded, flexible broken rod‐like conformation which is clearly distinguishable from the “molten globule” state. The stability of both N and H states depends on pH and methanol concentration. Thus a phase diagram involving N and H was constructed.
Serial increase in the thermal stability of 3‐isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution
Authors: Akanuma Satoshi , Yamagishi Akihiko , Oshima Tairo , Tanaka Nobuo
Published: July 2010

We improved the thermal stability of 3‐isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3‐isopropylmalate dehydrogenase was integrated into the chromosome of a leuB‐deficient strain of T. thermophilus. The resulting transformant showed a leucine‐autotrophy at 56°C but not at 61°C and above. Phenotypically thermostabilized strains that can grow at 61°C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70°C and mutants that showed a leucine autotrophic growth at 70°C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine‐308 to isoleucine, isoleucine‐95 to leucine, and methionine‐292 to isoleucine. The mutant enzymes with these amino acidreplacements were more stable against heat treatment than the wild‐type enzyme. Furthermore, the triple‐mutant enzyme showed significantly higher specific activity than that of the wild‐type enzyme.
Motional dynamics of residues in a β‐hairpin peptide measured by 13C‐NMR relaxation
Authors: Ramirez‐Alvarado Marnia , Dargan Vladimir A. , Mayo Kevin H. , Serrano Luis
Published: July 2010

Structurally characterizing partially folded peptides is problematic given the nature of their transient conformational states. 13C‐NMR relaxation data can provide information on the geometry of bond rotations, motional restrictions, and correlated bond rotations of the backbone and side chains and, therefore, is one approach that is useful to assess the presence of folded structure within a conformational ensemble. A peptide 12mer, R1GITVNG7KTYGR12, has been shown to partially fold in a relatively stable β‐hairpin conformation centered at NG. Here, five residues, G2, V5, G7, Y10, G1l, were selectively 13C‐enriched, and13C‐NMR relaxation experiments were performed to obtain auto‐ and cross‐correlation motional order parameters, correlation times, bond rotation angular variances, and bond rotational correlation coefficients. Our results indicate that, of the three glycines, G7 within the hairpin β‐turn displays the most correlated ϕ(t),Ψ(t) rotations with its axis of rotation bisecting the angle defined by the H‐C‐H bonds. These positively correlated bond rotations give rise to “twisting” type motions of the HCH group. V5 and Y10 ϕ,Ψ bond rotations are also positively correlated, with their C#bTCαH groups undergoing similar “twisting” type motions. Motions of near‐terminal residues G2 and G1l are less restricted and less correlated and are best described as wobbling‐in‐a‐cone. V5 and Y10 side‐chain motions, aside from being highly restricted, were found to be correlated with ϕ,Ψ bond rotations. At 303 K, where the hairpin is considered “unfolded,” the peptide exists in a transient, collapsed state because backbone and side‐chain motions of V5, G7, and Y10 remain relatively restricted, unlike their counterparts in GXG‐based tripeptides. These results provide unique information toward understanding conformational variability in the unfolded state of proteins, which is necessary to solve the protein folding problem.
Crystal structure of glycosylasparaginase from Flavobacterium meningosepticum
Authors: Xuan Jiancheng , Tarentino Anthony L. , Grimwood Brian G. , Plummer Thomas H. , Roey Patrick Van , Cui Tao , Guan Chudi
Published: July 2010

The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 Å resolution. This enzyme is a glycoamidase that cleaves the link between the asparagine and the N‐acetylglucosamine of N‐linked oligosaccharides and plays a major role in the degradation of glycoproteins. The three‐dimensional structure of the bacterial enzyme is very similar to that of the human enzyme, although it lacks the four disulfide bridges found in the human enzyme. The main difference is the absence of a small random coil domain at the end of the α‐chain that forms part of the substrate binding cleft and that has a role in the stabilization of the tetramer of the human enzyme. The bacterial glycosylasparaginase is observed as an (αβ)2‐tetramer in the crystal, despite being a dimer in solution. The study of the structure of the bacterial enzyme allows further evaluation of the effects of disease‐causing mutations in the human enzyme and confirms the suitability of the bacterial enzyme as a model for functional analysis.