bluuebunny/crossref_metadata_2025 huggingface dataset:
Interlobe communication in multiple calcium‐binding site mutants of Drosophila calmodulin
Authors: Mukherjea Poushali , Maune John F. , Beckingham Kathy
Published: July 2010

We have generated mutants of Drosophila calmodulin in which pairs of calcium‐binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the —Z position glutamate residue with glutamine. Mutants inactivated in both N‐terminal sites (B12Q) or both C‐terminal sites (B34Q), and two mutants with one N‐ and one C‐terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV‐difference spectroscopy and near‐UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr‐138) of the protein. These studies uncovered four situations in which Tyr‐138 in the C‐terminal lobe responds to a change in the calcium‐binding properties of the N‐terminal lobe. Further, they suggest that N‐terminal calcium‐binding events contribute strongly to the aberrant behavior of Tyr‐138 seen in mutants with a single functional C‐terminal calcium‐binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr‐138 produced by mutation of site 3 toward the wild‐type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK‐MLCK) established that all of the multiple binding site mutants are poor activators of SK‐MLCK. Thus, globally, the calcium‐induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr‐138 within the crystal structure of calmodulin suggests that effects of the N‐terminal lobe on this residue may be mediated via changes to the central linker region of the protein.
A stable intermediate in the thermal unfolding process of a chimeric 3‐isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes
Authors: Hayashi‐Iwasaki Yoko , Numata Koichi , Yamagishi Akihiko , Yutani Katsuhide , Sakurai Masahiro , Tanaka Nobuo , Oshima Tairo
Published: July 2010

The thermal unfolding process of a chimeric 3‐isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild‐type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration‐independent, whereas the second transition was protein concentration‐dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10–11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N‐ and C‐termini of the enzyme, and the second to that of domain 2 containing the subunit interface.
Crystallization and preliminary diffraction studies of NodL, a rhizobial O‐acetyl‐transferase involved in the host‐specific nodulation of legume roots
Authors: Dunn Steven M. , Moody Peter C. E. , Downie J. Allan , Shaw William V.
Published: July 2010

The NodL specified O‐acetyltransferase from the microbial symbiont Rhizobium leguminosarum has been over‐expressed in Escherichia coli and purified using affinity‐elution dye chromatography as the key step. The protein has been crystallized at 20 °C in 18% PEG 600, 0.1 M Tris/HCl buffer, pH 8.5, containing 1% dioxane, 0.25% octyl‐β‐glucoside, and 5 mM coenzyme A using the hanging drop vapor diffusion method. Ambient temperature X‐ray diffraction studies reveal the space group to be hexagonal (P6322) with lattice constants a = b = 77.08 å, c = 160.6 å, and α = β = 90°, γ = 120°. Crystals that are flash‐frozen to 120 K diffract beyond 2.7 å.
Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds
Authors: Gomar Jérôme , Petit Marie‐Christine , Sodano Patrick , Sy Denise , Marion Didier , Kader Jean‐Claude , Vovelle Françoise , Ptak Marius
Published: July 2010

The three‐dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1, 091 NOE‐derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (ϕ, ψ, χ1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C‐terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413–422) constitutes a new architecture for α‐class proteins. 1H NMR and fluorescence studies show that this protein forms well‐defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 ± 0.6 × 10−6 M and >10−3 M were obtained with lyso‐C16 and ‐C12, respectively. A structure model for a lipid—protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid—protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189–199).
Simulated annealing with restrained molecular dynamics using a flexible restraint potential: Theory and evaluation with simulated NMR constraints
Authors: Bassolino‐Klimas Donna , Tejero Roberto , Krystek Stanley R. , Metzler William J. , Montelione Gaetano T. , Bruccoleri Robert E.
Published: July 2010

A new functional representation of NMR‐derived distance constraints, the flexible restraint potential, has been implemented in the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137–168) for molecular structure generation. In addition, flat‐bottomed restraint potentials for representing dihedral angle and vicinal scalar coupling constraints have been introduced into CONGEN. An effective simulated annealing (SA) protocol that combines both weight annealing and temperature annealing is described. Calculations have been performed using ideal simulated NMR constraints, in order to evaluate the use of restrained molecular dynamics (MD) with these target functions as implemented in CONGEN. In this benchmark study, internuclear distance, dihedral angle, and vicinal coupling constant constraints were calculated from the energy‐minimized X‐ray crystal structure of the 46‐amino acid polypeptide crambin (1CRN). Three‐dimensional structures of crambin that satisfy these simulated NMR constraints were generated using restrained MD and SA. Polypeptide structures with extended backbone and side‐chain conformations were used as starting conformations. Dynamical annealing calculations using extended starting conformations and assignments of initial velocities taken randomly from a Maxwellian distribution were found to adequately sample the conformational space consistent with the constraints. These calculations also show that loosened internuclear constraints can allow molecules to overcome local minima in the search for a global minimum with respect to both the NMR‐derived constraints and conformational energy. This protocol and the modified version of the CONGEN program described here are shown to be reliable and robust, and are applicable generally for protein structure determination by dynamical simulated annealing using NMR data.
Thermosensitive mutants of the MPTP and hPTP1B protein tyrosine phosphatases: Isolation and structural analysis
Authors: Muise Eric S. , Vrielink Alice , Ennis Maurice A. , Lemieux Nancy H. , Tremblay Michel L.
Published: July 2010

A PCR‐based random mutagenesis procedure was employed to identify several thermosensitive mutants of the MPTP enzyme, the murine homologue of the human T‐cell PTPase and rat PTP‐S enzymes. Four mutants with varying degrees of thermosensitivity were characterized for their thermostability and refolding properties following incubation at the nonpermissive temperature. Structure analysis of these mutations based on the hPTP1B coordinate structure demonstrates a clear relationship between the position of each mutated amino acid relative to the catalytic cysteine residue and their thermostability. Introduction of two of these mutations in the related enzyme hPTP1B suggests that the structural defects and the resulting thermosensitivity of these mutations may represent an intrinsic property of all PTPase catalytic domains.
Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide‐specific phospholipase C δ1
Authors: Grobler Jay A. , Hurley James H.
Published: July 2010

Phosphatidylinositide‐specific phospholipase Cs (PI‐PLCs) catalyze the calcium‐dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI‐PLCs contain divergent regulatory regions, but all share three conserved regions: an N‐terminal pleckstrin homology (PH) domain, X, and Y. We report the high‐level expression and characterization of a recombinant “catalytic core” of rat PI‐PLC δ1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI‐PLC δδ1–134, is catalytically active versus phosphatidylinositol 4, 5‐bisphosphate in deoxycholate micelles with a Km of 182 μM and a Vmax of 27 μmol/min/mg. PI‐PLC δδ1–134 is monomeric and monodisperse as judged by dynamic light scattering. Far‐UV CD indicates a structure with approximately 35% α‐helix. A reversible change in the near‐ UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI‐PLC δδ1–134 in the absence of phospholipid. Triclinic crystals of PI‐PLC δδ1–134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/Å3 and on the self‐rotation function, there are two PI‐PLC δδ1–134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.
Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: Implications for domain switching
Authors: Murata Lauren B. , Schachman H. K.
Published: July 2010

Each catalytic (c) polypeptide chain of Escherichia coli aspartate transcarbamoylase (ATCase) is composed of two globular domains connected by two interdomain helices. Helix 12, near the C‐terminus, extends from the second domain back through the first domain, bringing the two termini close together. This helix is of critical importance for the assembly of a stable enzyme. The trimeric E. coli enzyme ornithine transcarbamoylase (OTCase) is proposed to be similar in tertiary and quaternary structure to the ATCase trimer and has a predicted α‐helical segment near its C‐terminus. In our companion paper, we have shown that this putative helix is essential for OTCase folding and assembly (Murata L, Schachman HK, 1996, Protein Sci 5:709–718). Here, the similarity between OTCase and the ATCase trimer, which are 32% identical in sequence, was tested further by the construction of several chimeras in which various structural elements were switched between the enzymes by genetic techniques. These elements included the two globular domains and regions containing the C‐terminal helices. In contrast to results reported previously (Houghton J, O'Donovan G, Wild J, 1989, Nature 338:172–174), none of the chimeric proteins exhibited in vivo activity and all were insoluble when overexpressed. Attempts to make hybrid trimers composed of c chains from ATCase and OTCase were also unsuccessful. These results underscore the complexities of specific intrachain and interchain side‐chain interactions required to maintain tertiary and quaternary structures in these enzymes.
4‐Oxalocrotonate tautomerase, a 41‐kDa homohexamer: Backbone and side‐chain resonance assignments, solution secondary structure, and location of active site residues by heteronuclear NMR spectroscopy
Authors: Stivers James T. , Abeygunawardana Chitrananda , Whitman Christian P. , Mildvan Albert S.
Published: July 2010

4‐Oxalocrotonate tautomerase (4‐OT), a homohexamer consisting of 62 residues per subunit, catalyzes the isomerization of unsaturated α‐keto acids using Pro‐1 as a general base (Stivers et al., 1996a, 1996b). We report the backbone and side‐chain 1H, 15N, and 13C NMR assignments and the solution secondary structure for 4‐OT using 2D and 3D homonuclear and heteronuclear NMR methods. The subunit secondary structure consists of an α‐helix (residues 13–30), two β‐strands (β1, residues 2–8; β2, residues 39–45), a β‐hairpin (residues 50–57), two loops (1, residues 9–12; II, 34–38), and two turns (I, residues 30–33; II, 47–50). The remaining residues form coils. The β1 strand is parallel to the β2 strand of the same subunit on the basis of cross strand NHi‐NHj NOEs in a 2D 15N‐edited 1H‐NOESY spectrum of hexameric 4‐OT containing two 15N‐labeled subunits/hexamer. The β1 strand is also antiparallel to another β1 strand from an adjacent subunit forming a subunit interface. Because only three such pairwise interactions are possible, the hexamer is a trimer of dimers. The diffusion constant, determined by dynamic light scattering, and the rotational correlation time (14.5 ns) estimated from 15N T1/T2 measurements, are consistent with the hexameric molecular weight of 41 kDa. Residue Phe‐50 is in the active site on the basis of transferred NOEs to the bound partial substrate 2‐oxo‐l, 6‐hexanedioate. Modification of the general base, Pro‐1, with the active site‐directed irreversible inhibitor, 3‐bromopyruvate, significantly alters the amide 15N and NH chemical shifts of residues in the β‐hairpin and in loop II, providing evidence that these regions change conformation when the active site is occupied.
Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: Leucine 66 to proline and leucine 91 to proline
Authors: Gray Terry M. , Arnoys Eric J. , Blankespoor Stephen , Born Tim , Jagar Rebekah , Everman Rebecca , Plowman Darla , Stair Angela , Zhang Daisy
Published: July 2010

A class of temperature‐sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 °C and no activity at 43 °C has been selected. These mutants, designated “tight” ts mutants, differ from most other T4 lysozyme mutants that are active at 43 °C, but only manifest their ts lesion by a reduced halo size around phage plaques after exposure of the growth plates to chloroform vapors. For example, in the series of T4 lysozyme mutants at position 157, the original randomly selected mutant, T157I, is the least stable of the series, yet, apart from the halo assay and subsequent in vitro protein stability measurements, this mutant is indistinguishable from wild type (WT) even at 43 °C.

Two mutants were identified: L91P and L66P. Both insert proline residues into α‐helical regions of the WT protein structure. The stabilities (δδG) as determined by urea denaturation are 8.2 kcal/mol for L91P and 7.1 kcal/mol for L66P. CD spectra indicate that no major conformational changes have occurred in the mutant structures. The structures of the mutants were modeled with a 40‐ps molecular dynamics simulation using explicit solvent. For L91P, the reduction of stability appears to be due to an unsatisfied hydrogen bond in the α‐helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain α‐helix, at least two unsatisfied hydrogen bonds, and a newly formed solvent‐filled pocket that protrudes into the hydrophobic core, possibly reducing the stabilizing contribution of a partially buried intrachain salt bridge.
The importance of larger data sets for protein secondary structure prediction with neural networks
Authors: Chandonia John‐Marc , Karplus Martin
Published: July 2010

A neural network algorithm is applied to secondary structure and structural class prediction for a database of 318 nonhomologous protein chains. Significant improvement in accuracy is obtained as compared with performance on smaller databases. A systematic study of the effects of network topology shows that, for the larger database, better results are obtained with more units in the hidden layer. In a 32‐fold cross validated test, secondary structure prediction accuracy is 67.0%, relative to 62.6% obtained previously, without any evolutionary information on the sequence. Introduction of sequence profiles increases this value to 72.9%, suggesting that the two types of information are essentially independent. Tertiary structural class is predicted with 80.2% accuracy, relative to 73.9% obtained previously. The use of a larger database is facilitated by the introduction of a scaled conjugate gradient algorithm for optimizing the neural network. This algorithm is about 10–20 times as fast as the standard steepest descent algorithm.
Extremely thermostable L(+)‐lactate dehydrogenase from thermotoga maritima: Cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state
Authors: Ostendorp Ralf , Auerbach Günter , Jaenicke Rainer
Published: July 2010

L(+)‐lactate dehydrogenase (LDH; E.C.1.1.1.27) from the hyperthermophilic bacterium Thermotoga maritima has been shown to represent the most stable LDH isolated so far (Wrba A, Jaenicke R, Huber R, Stetter KO, 1990, Eur J Biochem 188:195–201). In order to obtain the enzyme in amounts sufficient for physical characterization, and to analyze the molecular basis of its intrinsic stability, the gene was cloned and expressed functionally in Escherichia coli. Growth of the cells and purification of the enzyme were performed aerobically at 26 °C, i.e., ca. 60° below the optimal growth temperature of Thermotoga. Two enzyme species with LDH activity were purified to homogeneity. Crystals of the enzyme obtained at 4 °C show satisfactory diffraction suitable for X‐ray analysis up to a resolution of 2.8 Å. As shown by gel‐permeation chromatography, chemical crosslinking, light scattering, analytical ultracentrifugation, and electron microscopy, the two LDH species represent homotetramers and homooctamers (i.e., dimers of tetramers), with a common subunit molecular mass of 35 kDa. The spectroscopic characteristics (UV absorption, fluorescence emission, near‐ and far‐UV CD) of the two species are indistinguishable. The calculated α‐helix content is 45%, in accordance with the result of homology modeling. Compared to the tetrameric enzyme, the octamer exhibits reduced specific activity, whereas KM is unaltered. The extreme intrinsic stability of the protein is reflected by its unaltered catalytic activity over 4 h at 85 °C; irreversible thermal denaturation becomes significant at ∼95 °C. The anomalous resistance toward chemical denaturation using guanidinium chloride and urea confirms this observation. Both the high optimal temperature and the pH optimum of the catalytic activity correspond to the growth conditions of T. maritima in its natural habitat.
Protein fold recognition using sequence‐derived predictions
Authors: Fischer Daniel , Eisenberg David
Published: July 2010

In protein fold recognition, one assigns a probe amino acid sequence of unknown structure to one of a library of target 3D structures. Correct assignment depends on effective scoring of the probe sequence for its compatibility with each of the target structures. Here we show that, in addition to the amino acid sequence of the probe, sequence‐derived properties of the probe sequence (such as the predicted secondary structure) are useful in fold assignment. The additional measure of compatibility between probe and target is the level of agreement between the predicted secondary structure of the probe and the known secondary structure of the target fold. That is, we recommend a sequence‐structure compatibility function that combines previously developed compatibility functions (such as the 3D‐1D scores of Bowie et al. [1991] or sequence‐sequence replacement tables) with the predicted secondary structure of the probe sequence.

The effect on fold assignment of adding predicted secondary structure is evaluated here by using a benchmark set of proteins (Fischer et al., 1996a). The 3D structures of the probe sequences of the benchmark are actually known, but are ignored by our method. The results show that the inclusion of the predicted secondary structure improves fold assignment by about 25%. The results also show that, if the true secondary structure of the probe were known, correct fold assignment would increase by an additional 8–32%. We conclude that incorporating sequence‐derived predictions significantly improves assignment of sequences to known 3D folds.

Finally, we apply the new method to assign folds to sequences in the SWISSPROT database; six fold assignments are given that are not detectable by standard sequence‐sequence comparison methods; for two of these, the fold is known from X‐ray crystallography and the fold assignment is correct.
Self‐consistently optimized statistical mechanical energy functions for sequence structure alignment
Authors: Koretke K. K. , Luthey‐Schulten Z. , Wolynes P. G.
Published: July 2010

A quantitative form of the principle of minimal frustration is used to obtain from a database analysis statistical mechanical energy functions and gap parameters for aligning sequences to three‐dimensional structures. The analysis that partially takes into account correlations in the energy landscape improves upon the previous approximations of Goldstein et al. (1994, 1995) (Goldstein R, Luthey‐Schulten Z, Wolynes P, 1994, Proceedings of the 27th Hawaii International Conference on System Sciences. Los Alamitos, California: IEEE Computer Society Press. pp 306–315; Goldstein R, Luthey‐Schulten Z, Wolynes P, 1995, In: Elber R, ed. New developments in theoretical studies of proteins. Singapore: World Scientific). The energy function allows for ordering of alignments based on the compatibility of a sequence to be in a given structure (i.e., lowest energy) and therefore removes the necessity of using percent identity or similarity as scoring parameters. The alignments produced by the energy function on distant homologues with low percent identity (less than 21%) are generally better than those generated with evolutionary information. The lowest energy alignment generated with the energy function for sequences containing prosite signatures but unknown structures is a structure containing the same prosite signature, providing a check on the robustness of the algorithm. Finally, the energy function can make use of known experimental evidence as constraints within the alignment algorithm to aid in finding the correct structural alignment.
Direct evidence for a two‐state protein unfolding transition from hydrogen‐deuterium exchange, mass spectrometry, and NMR
Authors: Yi Qian , Baker David
Published: July 2010

We use mass spectrometry in conjunction with hydrogen‐deuterium exchange and NMR to characterize the conformational dynamics of the 62‐residue IgG binding domain of protein L under conditions in which the native state is marginally stable. Mass spectra of protein L after short incubations in D2O reveal the presence of two distinct populations containing different numbers of protected protons. NMR experiments indicate that protons in the hydrophobic core are protected in one population, whereas all protons are exchanged for deuterons in the other. As the exchange period is increased, molecules are transferred from the former population to the latter. The absence of molecules with a subset of the core protons protected suggests that exchange occurs in part via a highly concerted transition to an excited state in which all protons exchange rapidly with deuterons. A steady increase in the molecular weight of the population with protected protons, and variation in the exchange rates of the individual protected protons indicates the presence of an additional exchange mechanism. A simple model in which exchange results from rapid (>105/s) local fluctuations around the native state superimposed upon transitions to an unfolded excited state at ∼0.06/s is supported by qualitative agreement between the observed mass spectra and the mass spectra simulated according to the model using NMR‐derived estimates of the proton exchange rates.
The heme redox center of chloroplast cytochrome f is linked to a buried five‐water chain
Authors: Martinez S. E. , Huang D. , Ponomarev M. , Cramer W. A. , Smith J. L.
Published: July 2010

The crystal structure of the 252‐residue lumen‐side domain of reduced cytochrome f, a subunit of the proton‐pumping integral cytochrome b6f complex of oxygenic photosynthetic membranes, was determined to a resolution of 1.96 Å from crystals cooled to —35°. The model was refined to an R‐factor of 15.8% with a 0.013‐Å RMS deviation of bond lengths from ideality. Compared to the structure of cytochrome f at 20°, the structure at —35° has a small change in relative orientation of the two folding domains and significantly lower isotropic temperature factors for protein atoms. The structure revealed an L‐shaped array of five buried water molecules that extend in two directions from the NΔ1 of the heme ligand His 25. The longer branch extends 11 Å within the large domain, toward Lys 66 in the prominent basic patch at the top of the large domain, which has been implicated in the interaction with the electron acceptor, plastocyanin. The water sites are highly occupied, and their temperature factors are comparable to those of protein atoms. Virtually all residues that form hydrogen bonds with the water chain are invariant among 13 known cytochrome f sequences. The water chain has many features that optimize it as a proton wire, including insulation from the protein medium. It is suggested that this chain may function as the lumen‐side exit port for proton translocation by the cytochrome b6f complex.
The proteins encoded by the rbs operon of escherichia coli: II. Use of chimeric protein constructs to isolate and characterize RbsC
Authors: Zaitseva Jelena , Zhang Huide , Binnie R. Alan , Hermodson Mark
Published: July 2010

Chimeric genes encoding full‐length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly‐His leader peptide was incorporated so that Ni‐chelate affinity chromatography could be employed. The leader peptide and the bridge peptide were designed with factor Xa‐cleavable sites to permit recovery of the individual RbsA and RbsC protein. A rbsC gene encoding a poly‐His leader was also constructed and expressed. Both the chimeric RbsA‐C species and the poly‐HisRbsC were produced at levels that permitted isolation of the equivalent of milligram quantities of RbsC per liter of culture. This is a substantial increase in amounts from any previous RbsC production vectors. All proteins from the rbs operon have now been overproduced and substantially purified.
Secondary structure and NMR assignments ofbacillus circulansxylanase
Authors: Plesniak Leigh A. , Mcintosh Lawrence P. , Wakarchuk Warren W.
Published: July 2010

Bacillus circulansxylanase (BCX) is a member of the family of low molecular weight endo‐β‐(1,4)‐xylanases. The main‐chain1H,13C, and15N resonances of this 20.4‐kDa enzyme were assigned using heteronuclear NMR experiments recorded on a combination of selectively and uniformly labeled protein samples. Using chemical shift, NOE, J coupling, and amide hydrogen exchange information, 14 β‐strands, arranged in a network of three β‐sheets, and a single α‐helix were identified in BCX. The NMR‐derived secondary structure and β‐sheet topology agree closely with that observed in the crystal structure of this protein. The HNof Ile 118 has a strongly upfield‐shifted resonance at 4.03 ppm, indicative of a potential amide‐aromatic hydrogen bond to the indole ring of Trp 71. This interaction, which is conserved in all low molecular weight xylanases of known structure, may play an important role in establishing the active site conformation of these enzymes. Following hen egg white and bacteriophage T4 lysozymes,B. circulansxylanase represents the third family of β‐glycanases for which extensive NMR assignments have been reported. These assignments provide the background for detailed studies of the mechanism of carbohydrate recognition and hydrolysis by this bacterial xylanase.
In vitro methionine oxidation of escherichia coli‐derived human stem cell factor: Effects on the molecular structure, biological activity, and dimerization
Authors: Hsu Yueh‐Rong , Narhi Linda O. , Spahr Christopher , Langley Keith E. , Lu Hsieng S.
Published: July 2010

The effect of oxidation of the methionine residues of Escherichia coli‐derived recombinant human stem cell factor (huSCF) to methionine sulfoxide on the structure and activity of SCF was examined. Oxidation was performed using hydrogen peroxide under acidic conditions (pH 5.0). The kinetics of oxidation of the individual methionine residues was determined by quantitation of oxidized and unoxidized methionine‐containing peptides, using RP‐HPLC of Asp‐N endoproteinase digests. The initial oxidation rates for Met159, Met−1, Met27, Met36, and Met48 were 0.11 min−1, 0.098 min−1, 0.033 min−1 0.0063 min−1, and 0.00035 min−1, respectively, when SCF was incubated in 0.5% H2O2 at room temperature. Although oxidation of these methionines does not affect the secondary structure of SCF, the oxidation of Met36 and Met48 affects the local structure as indicated by CD and fluorescence spectroscopy. The 295‐nm Trp peak in the near‐UV CD is decreased upon oxidation of Met36, and lost completely following the oxidation of Met48, indicating that the Trp44 environment is becoming significantly less rigid than it is in native SCF. Consistent with this result, the fluorescence spectra revealed that Trp44 becomes more solvent exposed as the methionines are oxidized, with the hydrophobicity of the Trp44 environment decreasing significantly. The oxidations of Met36 and Met48 decrease biological activity by 40% and 60%, respectively, while increasing the dissociation rate constant of SCF dimer by two‐ and threefold. These results imply that the oxidation of Met36 and Met48 affects SCF dimerization and tertiary structure, and decreases biological activity.
Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences
Authors: Gryk Michael R. , Jardetzky Oleg , Klig Lisa S. , Yanofsky Charles
Published: July 2010

Trp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA‐binding domains of both the wild‐type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA‐binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild‐type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild‐type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.
Crystallization of the Aα subunit of protein phosphatase 2A
Authors: Skidmore Michael O. , Walter Gernot , Sawaya Michael R. , Everse Stephen J. , Parkin Sean , Rupp Bernhard , Hope Håkon
Published: July 2010

The Aα subunit of human protein phosphatase 2A forms crystals in space group P21 with cell dimensions a = 104.0, b = 174.9, c = 168.2 Å, and β angle = 90.2°. At cryogenic temperatures, the crystals diffracted to a resolution limit of ∼ 3.0 Å. Based on the unit cell dimensions and a calculated molecular mass of 65,277 Da, the Matthews coefficient suggests eight molecules per asymmetric unit. Two native data sets were collected to a nominal resolution of 3.0 Å and merged to provide a set that is 93% complete, with Rsvm of 9.9%.
Hydrophobic folding units derived from dissimilar monomer structures and their interactions
Authors: Tsai Chung‐Jung , Nussinov Ruth
Published: July 2010

We have designed an automated procedure to cut a protein into compact hydrophobic folding units. The hydrophobic units are large enough to contain tertiary non‐local interactions, reflecting potential nucleation sites during protein folding. The quality of a hydrophobic folding unit is evaluated by four criteria. The first two correspond to visual characterization of a structural domain, namely, compactness and extent of isolation. We use the definition of Zehfus and Rose (Zehfus MH, Rose GD, 1986, Biochemistry 25:335–340) to calculate the compactness of a cut protein unit. The isolation of a unit is based on the solvent accessible surface area (ASA) originally buried in the interior and exposed to the solvent after cutting. The third quantity is the hydrophobicity, equivalent to the fraction of the buried non‐poiar ASA with respect to the total non‐polar ASA. The last criterion in the evaluation of a folding unit is the number of segments it includes. To conform with the rationale of obtaining hydrophobic units, which may relate to early folding events, the hydrophobic interactions are implicitly and explicitly applied in their generation and assessment. We follow Holm and Sander (Holm L, Sander C, 1994, Proteins 79:256–268) to reduce the multiple cutting‐point problem to a one‐dimensional search for all reasonable trial cuts. However, as here we focus on the hydrophobic cores, the contact matrix used to obtain the first non‐trivial eigenvector contains only hydrophobic contacts, rather than all, hydrophobic and hydrophilic, interactions. This dataset of hydrophobic folding units, derived from structurally dissimilar single chain monomers, is particularly useful for investigations of the mechanism of protein folding. For cases where there are kinetic data, the one or more hydrophobic folding units generated for a protein correlate with the two or with the three‐state folding process observed. We carry out extensive amino acid sequence order independent structural comparisons to generate a structurally non‐redundant set of hydrophobic folding units for fold recognition and for statistical purposes.
Probing the active site of α‐class rat liver glutathione S‐transferases using affinity labeling by monobromobimane
Authors: Hu Longqin , Borleske Barbara L. , Colman Roberta F.
Published: July 2010

Monobromobimane (mBBr) is a substrate of both μ‐ and α‐class rat liver glutathione S‐transferases, with Km values of 0.63 μM and 4.9 μM for the μ‐class isozymes 3–3 and 4–4, respectively, and 26 μM for the α‐class isozymes 1–1 and 2–2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1–1 as well as μ‐class isozymes, but not of the α‐class 2–2 isozyme. Incubation of rat liver isozyme 1–1 with mBBr at pH 7.5 and 25 °C results in a time‐dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the μ‐class isozyme 3–3 and 4–4. The rate of inactivation of 1–1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S‐methyl glutathione. In contrast, 17β‐estradiol‐3,17‐disulfate (500 μM) gives a 12.5‐fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1–1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17β‐estradiol‐3,17‐disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1–1 isozyme, the other α‐class isozyme (2–2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S‐transferase isozymes.
Studies of protein‐protein interfaces: A statistical analysis of the hydrophobic effect
Authors: Tsai Chung‐Jung , Lin Shuo Liang , Wolfson Haim J. , Nussinov Ruth
Published: July 2010

Data sets of 362 structurally nonredundant protein‐protein interfaces and of 57 symmetry‐related oligomeric interfaces have been used to explore whether the hydrophobic effect that guides protein folding is also the main driving force for protein‐protein associations. The buried nonpolar surface area has been used to measure the hydrophobic effect. Our analysis indicates that, although the hydrophobic effect plays a dominant role in protein‐protein binding, it is not as strong as that observed in the interior of protein monomers. Comparison of the interiors of the monomers with those of the interfaces reveals that, in general, the hydrophobic amino acids are more frequent in the interior of the monomers than in the interior of the protein‐protein interfaces. On the other hand, a higher proportion of charged and polar residues are buried at the interfaces, suggesting that hydrogen bonds and ion pairs contribute more to the stability of protein binding than to that of protein folding. Moreover, comparison of the interior of the interfaces to protein surfaces indicates that the interfaces are poorer in polar/charged than the surfaces and are richer in hydrophobic residues. The interior of the interfaces appears to constitute a compromise between the stabilization contributed by the hydrophobic effect on the one hand and avoiding patches on the protein surfaces that are too hydrophobic on the other. Such patches would be unfavorable for the unassociated monomers in solution. We conclude that, although the types of interactions are similar between protein‐protein interfaces and single‐chain proteins overall, the contribution of the hydrophobic effect to protein‐protein associations is not as strong as to protein folding. This implies that packing patterns and interatom, or interresidue, pairwise potential functions, derived from monomers, are not ideally suited to predicting and assessing ligand associations or design. These would perform adequately only in cases where the hydrophobic effect at the binding site is substantial.
Thermodynamics of maltose binding protein unfolding
Authors: Novokhatny Valery , Ingham Kenneth
Published: July 2010

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto‐oligosaccharides. It is used widely as a carrier protein for the production of recombinant fusion proteins. The melting of recombinant MBP was studied by differential scanning and titration calorimetry and fluorescence spectroscopy under different solvent conditions. MBP exhibits a single peak of heat absorption with a ΔHcal/ΔHvH ratio in the range of 1.3–1.5, suggesting that the protein comprises two strongly interacting thermodynamic domains. Binding of maltose resulted in elevation of the TM by 8–15 °C, depending on pH. The presence of ligand at neutral pH, in addition to shifting the melting process to higher temperature, caused it to become more cooperative. The ΔHcal/ΔHvH ratio decreased to unity, indicating that the two domains melt together in a single two‐state transition. This ligand‐induced merging of the two domains appears to occur only at neutral pH, because at low pH maltose simply stabilized MBP and did not cause a decrease of the ΔHcal/ΔH
vH ratio. Binding of maltose to MBP is characterized by very low enthalpy changes,∼‐1 kcal/mol. The melting of MBP is accompanied by an exceptionally large change in heat capacity, 0.16 cal/K‐g, which is consistent with the high amount of nonpolar surface—0.72 Å2/g—that becomes accessible to solvent in the unfolded state. The high value of ΔCP determines a very steep ΔG versus T profile for this protein and predicts that cold denaturation should occur above freezing temperatures. Evidence for this was provided by changes in fluorescence intensity upon cooling the protein. A sigmoidal cooperative transition with a midpoint near 5°C was observed when MBP was cooled at low pH. Analysis of the melting of several fusion proteins containing MBP illustrated the feasibility of assessing the folding integrity of recombinant products prior to separating them from the MBP carrier protein.
Structures of N‐termini of helices in proteins
Authors: Doig Andrew J. , Stapley Benjamin J. , Macarthur Malcolm W. , Thornton Janet M.
Published: July 2010

We have surveyed 393 N‐termini of α‐helices and 156 N‐termini of 310‐helices in 85 high resolution, non‐homologous protein crystal structures for N‐cap side‐chain rotamer preferences, hydrogen bonding patterns, and solvent accessibilities. We find very strong rotamer preferences that are unique to N‐cap sites. The following rules are generally observed for N‐capping in α‐helices: Thr and Ser N‐cap side chains adopt the gauche— rotamer, hydrogen bond to the N3 NH and have Ψ restricted to 164 ± 8°. Asp and Asn N‐cap side chains either adopt the gauche— rotamer and hydrogen bond to the N3 NH with Ψ = 172 ± 10°, or adopt the trans rotamer and hydrogen bond to both the N2 and N3 NH groups with Ψ = 107 ± 19°. With all other N‐caps, the side chain is found in the gauche + rotamer so that the side chain does not interact unfavorably with the N‐terminus by blocking solvation and Ψ is unrestricted. An i, i + 3 hydrogen bond from N3 NH to the N‐cap backbone C=O in more likely to form at the N‐terminus when an unfavorable N‐cap is present. In the 310‐helix Asn and Asp remain favorable N‐caps as they can hydrogen bond to the N2 NH while in the trans rotamer; in contrast, Ser and Thr are disfavored as their preferred hydrogen bonding partner (N3 NH) is inaccessible. This suggests that Ser is the optimum choice of N‐cap when α‐helix formation is to be encouraged while 310‐helix formation discouraged. The strong energetic and structural preferences found for N‐caps, which differ greatly from positions within helix interiors, suggest that N‐caps should be treated explicitly in any consideration of helical structure in peptides or proteins.
Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: Secondary structure propensities are not conserved in proteins with the same fold
Authors: Ramirez‐Alvarado Marina , Serrano Luis , Blanco Francisco J.
Published: July 2010

The solution conformation of three peptides corresponding to the two β‐hairpins and the α‐helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non‐native locally folded structures, but no well‐defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the α‐helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non‐helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native‐like secondary structure propensities, although a minimum tendency to avoid non‐native structures and to favor native ones could be required.
LPFC: An internet library of protein family core structures
Authors: Schmidt Robert , Altman Russ B. , Gerstein Mark
Published: July 2010

As the number of protein molecules with known, high‐resolution structures increases, it becomes necessary to organize these structures for rapid retrieval, comparison, and analysis. The Protein Data Bank (PDB) currently contains nearly 5,000 entries and is growing exponentially. Most new structures are similar structurally to ones reported previously and can be grouped into families. As the number of members in each family increases, it becomes possible to summarize, statistically, the commonalities and differences within each family. We reported previously a method for finding the atoms in a family alignment that have low spatial variance and those that have higher spatial variance (i.e., the “core” atoms that have the same relative position in all family members and the “non‐core” atoms that do not). The core structures we compute have biological significance and provide an excellent quantitative and visual summary of a multiple structural alignment. In order to extend their utility, we have constructed a library of protein family cores, accessible over the World Wide Web at http://www‐smi.stanford.edu/projects/helix/LPFC/. This library is generated automatically with publicly available computer programs requiring only a set of multiple alignments as input. It contains quantitative analysis of the spatial variation of atoms within each protein family, the coordinates of the average core structures derived from the families, and display files (in bitmap and VRML formats). Here, we describe the resource and illustrate its applicability by comparing three multiple alignments of the globin family. These three alignments are found to be similar, but with some significant differences related to the diversity of family members and the specific method used for alignment.