bluuebunny/crossref_metadata_2025 huggingface dataset:
Design and structural analysis of an engineered thermostable chicken lysozyme
Authors: Shih Phoebe , Kirsch Jack F.
Published: February 2009

A hyperstable (hs) variant of chicken egg‐white lysozyme with enhanced thermal (ΔTm ≈︁ +10.5 °C) and chemical (ΔCm for guanidine hydrochloride denaturation = +1.3 M) stabilities relative to wild‐type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 °C) to 4.0 (differential scanning calorimetry, 74 °C) kcal mol−1 greater than that of WT. The specific activity of the hs variant is 2.5‐fold greater than that of WT. The choice of mutations came from diverse sources: (1) The I55L/S91T core construct with ΔTm = 3.3 °C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050–2062). (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 31 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The D101S variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.
Characterization of the isolated cAMP‐binding B domain of cAMP‐dependent protein kinase
Authors: Shabb John B. , Kapphahn Mark A. , Muhonen Wallace M. , Poteet Celeste E. , Baker Nancy E. , Corbin Jackie D.
Published: February 2009

A 14.4‐kDa cAMP‐binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP‐dependent protein kinase type Iα regulatory subunit (RIα). The full‐length RIα from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fragment was Ser‐252, indicating that it encompassed the entire predicted B domain. Although the [3H]cAMP and [3H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RIα, the [3H]cAMP exchange rate was comparable to that of the B domain of full‐length RIα containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high‐affinity cAMP‐binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2‐mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP‐binding domain can function independently of any other domain structures of the R subunit.
Are proteins ideal mixtures of amino acids? Analysis of energy parameter sets
Authors: Godzik Adam , Skolnick Jeffrey , Koliński Andrzej
Published: February 2009

Various existing derivations of the effective potentials of mean force for the two‐body interactions between amino acid side chains in proteins are reviewed and compared to each other. The differences between different parameter sets can be traced to the reference state used to define the zero of energy. Depending on the reference state, the transfer free energy or other pseudo‐one‐body contributions can be present to various extents in two‐body parameter sets. It is, however, possible to compare various derivations directly by concentrating on the “excess” energy— a term that describes the difference between a real protein and an ideal solution of amino acids. Furthermore, the number of protein structures available for analysis allows one to check the consistency of the derivation and the errors by comparing parameters derived from various subsets of the whole database. It is shown that pair interaction preferences are very consistent throughout the database. Independently derived parameter sets have correlation coefficients on the order of 0.8, with the mean difference between equivalent entries of 0.1kT. Also, the low‐quality (low resolution, little or no refinement) structures show similar regularities. There are, however, large differences between interaction parameters derived on the basis of crystallographic structures and structures obtained by the NMR refinement. The origin of the latter difference is not yet understood.
Cα‐based torsion angles: A simple tool to analyze protein conformational changes
Authors: Flocco Maria M. , Mowbray Sherry L.
Published: February 2009

A simple method is presented for the analysis of protein conformational changes based on the comparison of torsion angles defined by four consecutive Cα atoms. The technique was applied successfully to proteins that undergo hinge motion and shear motion. In the case of both MBP and LAO, which represent examples of hinge motion, the plot of the differences in Cα‐torsion angles between the open and closed forms of the proteins helped us to formulate a more thorough description of the conformational change: a large displacement of one domain with respect to the other where one of the domains does not behave like a rigid body but exhibits some degree of flexibility. The analysis of citrate synthase, which is an example of shear motion, shows that the largest differences in Cα‐torsion angles between the open and closed conformations are clustered around residues that belong to segments connecting α‐helices, whereas the helices themselves appear to be rigid; this is in agreement with previous results obtained by detailed least‐squares superpositions (Lesk AM, Chothia C, 1984, J Mol Biol 174:175–191).
Relative effectiveness of various anions on the solubility of acidicHypoderma lineatumcollagenase at pH 7.2
Authors: Carbonnaux Claire , Ries‐Kautt Madeleine , Ducruix Arnaud
Published: February 2009

The effects of various anions on decreasing the solubility of acidicHypoderma lineatumcollagenase at pH 7.2 and 18 °C were qualitatively defined by replacing the crystallizing agent of known crystallization conditions by various ammonium salts. The solubility curves measured in the presence of the sulfate, phosphate, citrate, and chloride ammonium salts gave the following ranking of anions: HPO42‐/H2PO4−> SO42‐> citrate3‐/citrate2‐>> Cl−. This order is in agreement with the Hofmeister series. In a previous study on the solubility at pH 4.5 of lysozyme, a basic protein, the effectiveness of anions in decreasing the solubility was found to be in the reverse order. This suggests that the effectiveness of anions in the crystallization of proteins is dependent on the net charge of the protein, i.e., depending on whether a basic protein is crystallized at acidic pH or an acidic protein at basic pH.
Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding
Authors: Myers Jeffrey K. , Nick Pace C. , Martin Scholtz J.
Published: February 2009

Denaturant m values, the dependence of the free energy of unfolding on denaturant concentration, have been collected for a large set of proteins. The m value correlates very strongly with the amount of protein surface exposed to solvent upon unfolding, with linear correlation coefficients of R = 0.84 for urea and R = 0.87 for guanidine hydrochloride. These correlations improve to R = 0.90 when the effect of disulfide bonds on the accessible area of the unfolded protein is included. A similar dependence on accessible surface area has been found previously for the heat capacity change (ΔCp), which is confirmed here for our set of proteins. Denaturant m values and heat capacity changes also correlate well with each other. For proteins that undergo a simple two‐state unfolding mechanism, the amount of surface exposed to solvent upon unfolding is a main structural determinant for both m values and ΔCp.
The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase
Authors: Dean Antony M. , Dvorak Laura
Published: February 2009

The kinetic mechanism of the oxidative decarboxylation of 2R,3S‐isopropylmalate by the NAD‐dependent isopropylmalate dehydrogenase of Thermus thermophilus was investigated. Initial rate results typical of random or steady‐state ordered sequential mechanisms are obtained for both the wild‐type and two mutant enzymes (E87G and E87Q) regardless of whether natural or alternative substrates (2R‐malate, 2R,3S‐tartrate and/or NADP) are utilized. Initial rate data fail to converge on a rapid equilibrium‐ordered pattern despite marked reductions in specificity (kcat/Km) caused by the mutations and alternative substrates. Although the inhibition studies alone might suggest an ordered kinetic mechanism with cofactor binding first, a detailed analysis reveals that the expected noncompetitive patterns appear uncompetitive because the dissociation constants from the ternary complexes are far smaller than those from the binary complexes. Equilibrium fluorescence studies both confirm the random binding of substrates and the kinetic estimates of the dissociation constants of the substrates from the binary complexes. The latter are not disturbed markedly by the mutations at site 87. Mutations at site 87 do not affect the dissociation constants from the binary complexes, but do greatly increase the Michaelis constants, indicating that E87 helps stabilize the Michaelis complex of the wild‐type enzyme. The available structural data, the patterns of the kinetics results, and the structure of a pseudo‐Michaelis complex of the homologous isocitrate dehydrogenase of Escherichia coli suggest that E87 interacts with the nicotinamide ring.
Molecular modeling studies of HIV‐1 reverse transcriptase nonnucleoside inhibitors: Total energy of complexation as a predictor of drug placement and activity
Authors: Kroeger Smith Marilyn B. , Hughes Stephen H. , Boyer Paul L. , Michejda Christopher J. , Rouzer Carol A. , Taneyhill Lisa A. , Smith Richard H. , Smith Nathan A. , Janssen Paul A.J. , Moereels Henri , Koymans Luc , Arnold Edward , Ding Jianping , Das Kalyan , Zhang Wanyi
Published: February 2009

Computer modeling studies have been carried out on three nonnucleoside inhibitors complexed with human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT), using crystal coordinate data from a subset of the protein surrounding the binding pocket region. Results from the minimizations of solvated complexes of 2‐cyclopropyl‐4‐methyl‐5,11‐dihydro‐5H‐dipyrido[3,2‐b:2′,3′‐e][1,4]diazepin‐6‐one (nevirapine), α‐anilino‐2, 6‐dibromophenylacetamide (α‐APA), and 8‐chloro‐tetrahydro‐imidazo(4,5,1‐jk)(1,4)‐benzodiazepin‐2(1H)‐thione (TIBO) show that all three inhibitors maintain a very similar conformational shape, roughly overlay each other in the binding pocket, and appear to function as π‐electron donors to aromatic side‐chain residues surrounding the pocket. However, side‐chain residues adapt to each bound inhibitor in a highly specific manner, closing down around the surface of the drug to make tight van der Waals contacts. Consequently, the results from the calculated minimizations reveal that only when the inhibitors are modeled in a site constructed from coordinate data obtained from their particular RT complex can the calculated binding energies be relied upon to predict the correct orientation of the drug in the pocket. In the correct site, these binding energies correlate with EC50values determined for all three inhibitors in our laboratory. Analysis of the components of the binding energy reveals that, for all three inhibitors, solvation of the drug is endothermic, but solvation of the protein is exothermic, and the sum favors complex formation. In general, the protein is energetically more stable and the drug less stable in their complexes as compared to the reactant conformations. For all three inhibitors, interaction with the protein in the complex is highly favorable. Interactions of the inhibitors with individual residues correlate with crystallographic and site‐specific mutational data. π‐Stacking interactions are important in binding and correlate with drug HOMO RHF/6–31G* energies. Modeling results are discussed with respect to the mechanism of complex formation and the design of nonnucleoside inhibitors that will be more effective against mutants of HIV‐1 RT that are resistant to the currently available drugs.
The Alacoil: A very tight, antiparallel coiled‐coil of helices
Authors: Gernert Kim M. , Surles Mark C. , Labean Thomas H. , Richardson Jane S. , Richardson David C.
Published: February 2009

The Alacoil is an antiparallel (rather than the usual parallel) coiled‐coil of α‐helices with Ala or another small residue in every seventh position, allowing a very close spacing of the helices (7.5–8.5 Å between local helix axes), often over four or five helical turns. It occurs in two distinct types that differ by which position of the heptad repeat is occupied by Ala and by whether the closest points on the backbone of the two helices are aligned or are offset by half a turn. The aligned, or ROP, type has Ala in position “d” of the heptad repeat, which occupies the “tip‐to‐tip” side of the helix contact where the Cα–Cβ bonds point toward each other. The more common offset, or ferritin, type of Alacoil has Ala in position “a” of the heptad repeat (where the Cα‐Cβ bonds lie back‐to‐back, on the “knuckle‐touch” side of the helix contact), and the backbones of the two helices are offset vertically by half a turn. In both forms, successive layers of contact have the Ala first on one and then on the other helix.

The Alacoil structure has much in common with the coiled‐coils of fibrous proteins or leucine zippers: both are α‐helical coiled‐coils, with a critical amino acid repeated every seven residues (the Leu or the Ala) and a secondary contact position in between. However, Leu zippers are between aligned, parallel helices (often identical, in dimers), whereas Alacoils are between antiparallel helices, usually offset, and much closer together. The Alacoil, then, could be considered as an “Ala anti‐zipper.” Leu zippers have a classic “knobs‐into‐holes” packing of the Leu side chain into a diamond of four residues on the opposite helix; for Alacoils, the helices are so close together that the Ala methyl group must choose one side of the diamond and pack inside a triangle of residues on the other helix.

We have used the ferritin‐type Alacoil as the basis for the de novo design of a 66‐residue, coiled helix hairpin called “Alacoilin.” Its sequence is: cmSPDQWDKE AAQYDAHAQE FEKKSHRNng TPEADQYRHM ASQY QAMAQK LKAIANQLKK Gseter (with “a” heptad positions underlined and nonhelical parts in lowercase), which we will produce and test for both stability and uniqueness of structure.
Covariation of residues in the homeodomain sequence family
Authors: Clarke Neil D.
Published: February 2009

Homeodomains are 60 amino acid DNA binding domains found in numerous eukaryotic transcription factors. The homeodomain family is a useful system for studying sequence‐structure relationships because several hundred sequences are known and the structures of several homeodomains have been determined. Covariation of amino acid residues in the homeodomain family has been investigated to see whether strongly covariant residue pairs can be understood in terms of the structure and function of these domains. Among 16 strongly covariant pairs examined, 2 are explained by the ability to form salt bridges, and 9 appear related to the DNA binding function of the proteins. For the remaining 5 pairs, the rationale for covariance remains unclear and the likelihood of artifactual correlations is discussed in the context of experimental and evolutionary biases in the selection of sequences. No significant correlation was found between covariance and structural proximity in the hydrophobic core.
Drosophilaengrailed‐1, 10‐phenanthroline chimeras as probes of homeodomain‐DNA complexes
Authors: Pan Clark Q. , Landgraf Ralf , Sigman David S.
Published: February 2009

We have converted theDrosophilaengrailed homeodomain into a sequence‐specific nuclease by linking the protein to the chemical nuclease 1,10‐phenanthroline‐copper (OP‐Cu). Unique cysteines were introduced at six positions into the homeodomain by site‐directed mutagenesis for the covalent attachment of OP‐Cu. The varied DNA‐binding affinity and specificity of these mutants and the DNA cleavage pattern of their OP‐Cu derivatives allowed us to assess the crystal structure of the engrailed homeodomain‐DNA complex. We have also achieved site‐specific double‐stranded DNA scission with one of the homeodomain mutants, E28C, which has the potential of being used to identify engrailed binding sites in the genome. Because the homeodomain is so well conserved among members of the homeodomain‐containing protein family, other homeodomain proteins can be converted into nucleases by attaching OP‐Cu at position 28 of their homeodomains.
Comparison of the accuracy of protein solution structures derived from conventional and network‐edited NOESY data
Authors: Hoogstraten Charles G. , Choe Soheui , Westler William M. , Markley John L.
Published: February 2009

Network‐editing experiments are variants of the basic NOESY experiment that allow more accurate direct measurement of interproton distances in macromolecules by defeating specific spin‐diffusion pathways. Two network‐editing approaches, block‐decoupled NOESY and complementary‐block‐decoupled‐NOESY, were applied as three‐dimensional, heteronuclear‐edited experiments to distance measurement in a small protein, turkey ovomucoid third domain (OMTKY3). Two‐hundred and twelve of the original 655 distance constraints observed in this molecule (Krezel AM et al., 1994, J Mol Biol 242:203–214) were improved by their replacement by distances derived from network‐edited spectra, and distance geometry/simulated annealing solution structure calculations were performed from both the unimproved and improved distance sets. The resulting two families of structures were found to differ significantly, the most important differences being the hinge angle of a β‐turn and an expansion of the sampled conformation space in the region of the reactive‐site loop. The structures calculated from network‐editing data are interpreted as a more accurate model of the solution conformation of OMTKY3.
The use of side‐chain packing methods in modeling bacteriophage repressor and cro proteins
Authors: Chung Su Yun , Subbiah S.
Published: February 2009

In recent years, it has been repeatedly demonstrated that the coordinates of the main‐chain atoms alone are sufficient to determine the side‐chain conformations of buried residues of compact proteins. Given a perfect backbone, the side‐chain packing method can predict the side‐chain conformations to an accuracy as high as 1.2 Å RMS deviation (RMSD) with greater than 80% of the χ angles correct. However, similarly rigorous studies have not been conducted to determine how well these apply, if at all, to the more important problem of homology modeling per se. Specifically, if the available backbone is imperfect, as expected for practical application of homology modeling, can packing constraints alone achieve sufficiently accurate predictions to be useful? Here, by systematically applying such methods to the pairwise modeling of two repressor and two cro proteins from the closely related bacteriophages 434 and P22, we find that when the backbone RMSD is 0.8 Å, the prediction on buried side chain is accurate with an RMS error of 1.8 Å and approximately 70% of the χ angles correctly predicted. When the backbone RMSD is larger, in the range of 1.6–1.8 Å, the prediction quality is still significantly better than random, with RMS error at 2.2 Å on the buried side chains and 60% accuracy on χ angles. Together these results suggest the following rules‐of‐thumb for homology modeling of buried side chains. When the sequence identity between the modeled sequence and the template sequence is >50% (or, equivalently, the expected backbone RMSD is <1 Å), side‐chain packing methods work well. When sequence identity is between 30–50%, reflecting a backbone RMS error of 1–2 Å, it is still valid to use side‐chain packing methods to predict the buried residues, albeit with care. When sequence identity is below 30% (or backbone RMS error greater than 2 Å), the backbone constraint alone is unlikely to produce useful models. Other methods, such as those involving the use of database fragments to reconstruct a template backbone, may be necessary as a complementary guide for modeling.
Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational change
Authors: Weitzman Cindy , Consler Thomas G. , Kaback H. Ronald
Published: February 2009

Six single‐Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp‐less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638–1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single‐Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog β,D‐galactopyranosyl 1‐thio‐β,D‐galactopyranoside (TDG). This effect was further characterized by site‐directed fluorescent studies with purified single‐Cys W33 → C permease labeled with 2‐(4′‐maleimidylanilino)‐naphthalene‐6‐sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5‐nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 μM. The effect of substrate binding on the rate of MIANS labeling of single‐Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS‐labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257–8263) that ligand binding induces a conformational change at the C‐terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.
Two steps in the transition between the native and acid states of bovine α‐lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?
Authors: Gussakovsky Eugene E. , Haas Elisha
Published: February 2009

A few studies indirectly support the existence of an intermediate in the transition of Ca2+‐saturated bovine α‐lactalbumin (α‐LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of α‐LA as well as the fluorescence of 8‐anilino‐1‐naphthalenesulfonic acid (ANS) bound to α‐LA. CPL of tryptophan and ANS was measured in the pH range of 2.5–6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, gem) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the gab, absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3–3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the α‐LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS gem showed an additional conformational transition at pH 4–5, which coincided with the pKa of Ca2+ dissociation (pKa 5), as predicted by Permyakov et al. (1981, Biochem Biophys Res Commun 100:191–197). The titration curve showed that there is a pH range between 3.7 and 4.1 in which α‐LA exists in an intermediate state between the N‐ and A‐state. We suggest that the intermediate is the premolten globule state characterized by a reduced Ca2+ binding to the α‐LA, native‐like tertiary structure, and reduced asymmetric fluctuation of the tertiary structure on the nanosecond time scale. This intermediate resembles the “critical activated state” theoretically deduced by Kuwajima et al. (1989, J Mol Biol 206:547–561). The present study demonstrates the power of CPL measurements for the investigation of folding/unfolding transitions in proteins.
Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis
Authors: Dunten Pete , Mowbray Sherry L.
Published: February 2009

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly‐Leu (glycyl‐L‐leucine) has been determined at 3.2 Å resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.
Irreversible thermal denaturation ofTorpedo californicaacetylcholinesterase
Authors: Kreimer David I. , Shnyrov Valery L. , Villar Enrique , Silman Israel , Weiner Lev
Published: February 2009

Thermal denaturation ofTorpedo californicaacetylcholinesterase, a disulfide‐linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two‐state kinetic scheme N → D, with activation energy 131 ± 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 ± 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol‐specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.
Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site
Authors: Liaw Shwu‐Huey , Kuo Ichun , Eisenberg David
Published: February 2009

Glutamine synthetase (GS) catalyzes the ATP‐dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel‐shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X‐ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50′, Ser 53′ of the adjacent sub‐unit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the γ‐glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 Å, between n1 and Tl+ is 4 Å, and between n2 and Tl+ is 7 Å. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the “side‐to‐side” intersubunit interaction through the cation‐protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50′ toward this monovalent cation site, essentially forming the site. This observation supports a two‐step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form γ‐glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the γ‐glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate γ‐glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.
Significance of structural changes in proteins: Expected errors in refined protein structures
Authors: Stroud Robert M. , Fauman Eric B.
Published: February 2009

A quantitative expression key to evaluating significant structural differences or induced shifts between any two protein structures is derived. Because crystallography leads to reports of a single (or sometimes dual) position for each atom, the significance of any structural change based on comparison of two structures depends critically on knowing the expected precision of each median atomic position reported, and on extracting it for each atom, from the information provided in the Protein Data Bank and in the publication. The differences between structures of protein molecules that should be identical, and that are normally distributed, indicating that they are not affected by crystal contacts, were analyzed with respect to many potential indicators of structure precision, so as to extract, essentially by “machine learning” principles, a generally applicable expression involving the highest correlates. Eighteen refined crystal structures from the Protein Data Bank, in which there are multiple molecules in the crystallographic asymmetric unit, were selected and compared. The thermal B factor, the connectivity of the atom, and the ratio of the number of reflections to the number of atoms used in refinement correlate best with the magnitude of the positional differences between regions of the structures that otherwise would be expected to be the same. These results are embodied in a six‐parameter equation that can be applied to any crystallographically refined structure to estimate the expected uncertainty in position of each atom. Structure change in a macromolecule can thus be referenced to the expected uncertainty in atomic position as reflected in the variance between otherwise identical structures with the observed values of correlated parameters.
How to measure and predict the molar absorption coefficient of a protein
Authors: Pace C. Nick , Vajdos Felix , Fee Lanette , Grimsley Gerald , Gray Theronica
Published: February 2009

The molar absorption coefficient, ε, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring ε for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319–326] and is based on data from Edelhoch [1967, Biochemistry 6:1948–1954].) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average ε values for these chromophores in a sample of 18 well‐characterized proteins have been estimated, and the ε values in water, propanol, 6 M guanidine hydrochloride (GdnHCl), and 8 M urea have been measured. For Trp, the average ε values for the proteins are less than the ε values measured in any of the solvents. For Tyr, the average ε values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured ε values for 80 proteins, the ε at 280 nm of a folded protein in water, ε(280), can best be predicted with this equation

ϵ(280) (M−1 cm−1) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125).

These ε(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict ε.
New protein functions in yeast chromosome VIII
Authors: Ouzounis Christos , Bork Peer , Casari Georg , Sander Chris
Published: February 2009

The analysis of the 269 open reading frames of yeast chromosome VIII by computational methods has yielded 24 new significant sequence similarities to proteins of known function. The resulting predicted functions include three particularly interesting cases of translation‐associated proteins: peptidyl‐tRNA hydrolase, a ribosome recycling factor homologue, and a protein similar to cytochrome b translational activator CBS2. The methodological limits of the meaningful transfer of functional information between distant homologues are discussed.
Structural similarities in the noncatalytic domains of phenylalanyl‐tRNA and biotin synthetases
Authors: Safro Mark , Mosyak Lidia
Published: February 2009

Detailed comparison between the structures of the Escherichia coli biotin synthetase/repressor protein (BirA) and the recently solved Thermus thermophilus phenylalanyl‐tRNA synthetase (PheRS) reveals significant similarities outside their respective catalytic domains. These comprise a DNA‐binding α+β domain and an Src‐homology 3 (SH3)‐like domain that were observed in both enzymes. This similarity provides a novel example in which all domains of one multidomain protein appear to be constituents of the other multidomain protein and supports a concept of a common ancestor for two different synthetase families.
Identification of the catalytic histidine residue participating in the charge‐relay system of carboxypeptidase Y
Authors: Jung Giman , Ueno Hiroshi , Hayashi Rikimaru , Liao Ta‐Hsiu
Published: February 2009

The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site‐specific reagent, a chloromethyl‐ketone derivative of benzyloxycarbonyl‐L‐phenylalanine. The single modified histidine residue was converted to Nτ ‐carboxymethyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic‐thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val‐Phe‐Asp‐Gly‐Gly‐cmHis‐MetO2‐Val‐Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X‐ray analysis to participate in the charge‐relay system of CPY.
Crystallization and preliminary X‐ray diffraction analysis of recombinant pentalenene synthase
Authors: Lesburg Charles A. , Lloyd Matthew D. , Cane David E. , Christianson David W.
Published: February 2009

Recombinant pentalenene synthase, a 42.5‐kDa sesquiterpene cyclase originally isolated from Streptomyces UC5319 and cloned in Escherichia coli, has been crystallized in space group P63 with unit cell dimensions a = b = 183.5 Å and c = 56.5 Å. Hexagonal prismatic crystals, approximately 0.2 × 0.2 × 0.3 mm, diffract to approximately 2.9 Å resolution using monochromatic synchrotron radiation. From the universal (and achiral) building block, farnesyl pyrophosphate, pentalenene synthase catalyzes the formation of four stereocenters in the construction of the three fused five‐membered rings of pentalenene; this novel sesquiterpene is a precursor to the pentalenolactone family of antibiotics.
3D domain swapping: A mechanism for oligomer assembly
Authors: Bennett Melanie J. , Schlunegger Michael P. , Eisenberg David
Published: February 2009

3D domain swapping is a mechanism for forming oligomeric proteins from their monomers. In 3D domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical protein chain. The result is an intertwined dimer or higher oligomer, with one domain of each subunit replaced by the identical domain from another subunit. The swapped “domain” can be as large as an entire tertiary globular domain, or as small as an α‐helix or a strand of a β‐sheet. Examples of 3D domain swapping are reviewed that suggest domain swapping can serve as a mechanism for functional interconversion between monomers and oligomers, and that domain swapping may serve as a mechanism for evolution of some oligomeric proteins. Domain‐swapped proteins present examples of a single protein chain folding into two distinct structures.
Phosphorylation of serine‐46 in HPr, a key regulatory protein in bacteria, results in stabilization of its solution structure
Authors: Pullen Katherine , Rajagopal Ponni , Klevit Rachel E. , Branchini Bruce R. , Reizer Jonathan , Saier Milton H. , Scholtz J. Martin , Huffine Mary Elizabeth
Published: February 2009

The serine‐phosphorylated form of histidine‐containing protein (HPr), a component of the phosphoenol‐pyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy and solvent denaturation studies. The results indicate that phosphorylation of Ser 46, the N‐cap of α‐helix‐B, does not cause a conformational change but rather stabilizes the helix. Amide proton exchange rates in helix‐B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaturation, with a ΔΔG of 0.7‐0.8 kcal mol−1. A mutant in which Ser 46 is replaced by aspartic acid shows a similar stabilization, indicating that an electrostatic interaction between the negatively charged groups and the helix macrodipole contributes significantly to the stabilization.
Comparison of atomic solvation parametric sets: Applicability and limitations in protein folding and binding
Authors: Juffer André H. , Eisenhaber Frank , Hubbard Simon J. , Walther Dirk , Argos Patrick
Published: February 2009

Atomic solvation parameters (ASP) are widely used to estimate the solvation contribution to the thermodynamic stability of proteins as well as the free energy of association for protein‐ligand complexes. They are also included in several molecular mechanics computer programs. In this work, a total of eight atomic solvation parametric sets has been employed to calculate the solvation contribution to the free energy of folding ΔGs for 17 proteins. A linear correlation between ΔGs and the number of residues in each protein was found for each ASP set. The calculations also revealed a great variety in the absolute value and in the sign of ΔGs values such that certain ASP sets predicted the unfolded state to be more stable than the folded, whereas others yield precisely the opposite. Further, the solvation contribution to the free energy of association of helix pairs and to the disassociation of loops (connection between secondary structural elements in proteins) from the protein tertiary structures were computed for each of the eight ASP sets and discrepancies were evident among them.
A simple and fast approach to prediction of protein secondary structure from multiply aligned sequences with accuracy above 70%
Authors: Mehta Perdeep K. , Heringa Jaap , Argos Patrick
Published: February 2009

To improve secondary structure predictions in protein sequences, the information residing in multiple sequence alignments of substituted but structurally related proteins is exploited. A database comprised of 70 protein families and a total of 2, 500 sequences, some of which were aligned by tertiary structural superpositions, was used to calculate residue exchange weight matrices within α‐helical, β‐strand, and coil substructures, respectively. Secondary structure predictions were made based on the observed residue substitutions in local regions of the multiple alignments and the largest possible associated exchange weights in each of the three matrix types. Comparison of the observed and predicted secondary structure on a per‐residue basis yielded a mean accuracy of 72.2%. Individual α‐helix, β‐strand, and coil states were respectively predicted at 66.4, 66.7, and 75.8°7o correctness, representing a well‐balanced three‐state prediction. The accuracy level, verified by cross‐validation through jack‐knife tests on all protein families, dropped, on average, to only 70.9%, indicating the rigor of the prediction procedure. On the basis of robustness, conceptual clarity, accuracy, and executable efficiency, the method has considerable advantage, especially with its sole reliance on amino acid substitutions within structurally related proteins.
Spectroscopic characterization of rhino viral protease 2a: Zn is essential for the structural integrity
Authors: Voss Tilman , Meyer Rainer , Sommergruber Wolfgang
Published: February 2009

Recently, protease 2A of human rhinovirus 2 (HRV2 2A) was shown to require a zinc ion for the formation of an active enzyme although zinc is not involved mechanistically. The data presented clearly show that the zinc ion bound to a picornaviral‐specific motif represents an essential component of the native structure, probably representing a new Zn‐binding motif. This structure, containing mostly β‐strand elements as shown by CD spectroscopy, changes drastically upon removal of zinc. The zinc‐depleted form does represent an intermediate with mostly unchanged secondary structure, but not a fully denatured random coil as obtained by guanidinium hydrochloride. This is indicated by the blue‐shifted fluorescence spectra and by CD. The native protein exhibited a cooperative phase transition at 53 °C. In contrast, the zinc‐depleted form did not show any transition at all, again demonstrating the stabilizing role of the zinc ion. A structural intermediate was observed during thermal and pH denaturation that may represent a molten globule, as suggested by its ANS binding.
Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes
Authors: Heyse Stephan , Vogel Horst , Sänger Michael , Sigrist Hans
Published: February 2009

A new method is presented for measuring sensitively the interactions between ligands and their membrane‐bound receptors in situ using integrated optics, thus avoiding the need for additional labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol, which can in principle be used with any glass‐like surface. In a first step, phospholipids carrying head‐group thiols were covalently immobilized onto SiO2‐TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N‐succinimidyl‐3‐maleimidopropionate, followed by the formation of thioethers between the surface‐grafted maleimides and the synthetic thiolipids. The surface‐attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self‐assembly from mixed lipid/detergent micelles. The step‐by‐step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous determination of optical thickness and one of the two refractive indices of the adsorbed organic layers. Surface coverages of 50‐60% were calculated for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45‐50 Å, which corresponds to the thickness of a fluid bilayer membrane.

Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid‐anchored receptor molecules into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated in the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. The specific antibody binding correlated well with the lipopeptide concentration in the outer monolayer.
The effects of filtration on protein nucleation in different growth media
Authors: Hirschler Joachim , Charon Marie‐Hélène , Fontecilla‐Camps Juan Carlos
Published: February 2009

Filtration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated. Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals. This decrease depended on the membrane cut‐off used for filtration. From this, the postulated factors aiding in nucleation are estimated to be 0.17 μm in diameter. The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results.

The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified. We did not find any time‐dependent change in the size or the number of crystals per drop.

Repeating the filtration experiments in agarose‐gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished. Far better crystallization results were obtained compared to ungelled media. However, there is a certain aging effect linked to filtration in gelled media. Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization. This suggests a secondary time‐dependent effect.