bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structure of the coat protein from the GA bacteriophage: Model of the unassembled dimer
Authors: Ni Chao‐Zhou , White Carol A. , Mitchell Richard S. , Wickersham John , Kodandapani Ramadurgam , Peabody David S. , Ely Kathryn R.
Published: February 2009

There are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 Å resolution (R‐factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L, 1993, J Mol Biol 234:620–639) or as an unassembled dimer (Ni CZ, Syed R, Kodandapani R, Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255–263). The structures differ in the FG loops and in the first turn of the αA helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA‐binding site located in an antiparallel β‐sheet across the dimer interface. Three differences involve residues in the center of this concave site; Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA‐binding specificity by GA or MS2 coat protein (Lim F, Spingola M, Peabody DS, 1994, J Biol Chem 269:9006–9010). This sequence difference reflects recognition of the nucleotide at position ‐5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.
Physicochemical basis for the rapid time‐action of LysB28ProB29‐insulin: Dissociation of a protein‐ligand complex
Authors: Bakaysa Diane L. , Radziuk Jerry , Havel Henry A. , Brader Mark L. , Li Shun , Dodd Steven W. , Beals John M. , Pekar Allen H. , Brems David N.
Published: February 2009

The rate‐limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self‐associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self‐associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m‐cresol and/or phenol) cause one such insulin analogue, LysB28ProB29‐human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand‐bound hexamer retains its rapid‐acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time‐action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer‐monomer interactions has been observed in the X‐ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995,Structure 3:615–622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt‐containing hexamers. These studies establish that rapid‐acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.
Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy
Authors: Reynaldo Luis P. , Villafranca Joseph J. , Horrocks William Dew.
Published: February 2009

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H20 and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 0.5 and 4.6 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor‐acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 1.5 Å. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single‐tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site nl was observed to decrease from Kd = 0.35 0.09 μM for GS0 to Kd = 0.06 0.02 μM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 0.9 μM and Kd = 2.6 0.7 μM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 0.02 μM for GS0 to Kd = 0.35 0.09 μM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 1.3 μM for GS0 and Kd = 4.0 1.0 μM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady‐state substrate experiments (Abell LM, Villafranca JJ, 1991. Biochemistry 30:1413–1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.
Cloning, expression, and crystallization of the Vδ domain of a human γδ T‐cell receptor
Authors: Lebedeva Marina I. , Fields Barry A. , Spits Hergen , Panchamoorthy Govindaswamy , Brenner Michael B. , Mariuzza Roy A.
Published: February 2009

T‐lymphocytes recognize a wide variety of antigens through highly diverse cell‐surface glycoproteins known as T‐cell receptors (TCRs). These disulfide‐linked heterodimers are composed of α and β or γ and δ polypeptide chains consisting of variable (V) and constant (C) domains non‐covalently associated with at least four invariant chains to form the TCR‐CD3 complex. It is well established that αβ TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three‐dimensional structure of αβ TCRs has recently become available through X‐ray crystallography. In contrast, the antigen specificity of γδ TCRs is much less well understood and their three‐dimensional structure is unknown. We have cloned the δ chain of a human TCR specific for the MHC class I HLA‐A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant Vδ domain was crystallized in a form suitable for X‐ray diffraction analysis. The crystals are orthorhombic, space group P21212 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 Å, and diffract to beyond 2.3 Å resolution. The ability of a Vδ domain produced in bacteria to form well‐ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of γδ TCRs.
Solvation effects are responsible for the reduced inhibitor affinity of some HIV‐1 PR mutants
Authors: Sussman Fredy , Villaverde M. Carmen , Davis Alan
Published: February 2009

The formulation of HIV‐1 PR inhibitors as anti‐viral drugs has been hindered by the appearance of protease strains that present drug resistance to these compounds. The mechanism by which the HIV‐1 PR mutants lower their affinity for the inhibitor is not yet fully understood. We have applied a modified Poisson‐Boltzmann method to the evaluation of the molecular interactions that contribute to the lowering of the inhibitor affinity to some polar mutants at position 82. These strains present drug resistance behavior and hence are ideally suited for these studies. Our results indicate that the reduction in binding affinity is due to the solvation effects that penalize the binding to the more polar mutants. The inhibitor binding ranking of the different mutants can be explained from the analysis of the different components of our free energy scoring function.
Design, synthesis, expression, and characterization of the genes for mouse FcγRIIb1 and FcγRIIb2 cytoplasmic regions
Authors: Chen Lixin , Thompson Nancy L. , Pielak Gary J.
Published: February 2009

The cytoplasmic regions of the mouse low‐affinity FcγRII isoforms, mFcγRIIb1, and mFcγRIIb2, play a key role in signal transduction by mediating different cellular functions. mFcγRIIb1 has a 94‐residue cytoplasmic region, whereas mFcγRIIb2 has a 47‐residue cytoplasmic region. Genes encoding the cytoplasmic regions of mFcγRIIb1 (b1‐94) and mFcγRIIb2 (b2‐47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli. A sequencespecific protease, thrombin, was used to release the b1‐94 peptide, which was purified by using HPLC. The b2‐47 peptide was synthesized chemically. CD spectropolarimetry was employed to examine the secondary structures of b1‐94 and b2‐47. These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature. The results indicate that the b1‐94 and b2‐47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant α‐helical structure.
Identification and structural influence of a differentially modified N‐terminal methionine in human S l00b
Authors: Smith Steven P. , Barber Kathryn R. , Shaw Gary S.
Published: February 2009

The calcium‐binding protein Sl00b is a homodimer comprised of two identical 91‐residue &subunits. Recombinant Sl00b is a heterogeneous protein, although the basis of this heterogeneity has not been established. We have used mass spectrometry and NMR spectroscopy to determine that heterogeneity in Sl00b arises from a mixture of formyl‐Sl00b and desformyl‐Sl00b when expressed in Escherichia coli. Reversed‐phase HPLC purification of these two forms of Sl00b has allowed the differences in N‐terminal composition to be used as a probe for tertiary contacts in the protein. The presence or absence of the N‐terminal formyl group affected the chemical shifts of sequence neighboring residues and those in the linker of the protein (residues 40‐43), indicating that these two regions are close in space.
Refined structures of three crystal forms of toxic shock syndrome toxin‐1 and of a tetramutant with reduced activity
Authors: Sridhar Prasad G. , Radhakrishnan Ramaswamy , Mitchell David T. , Earhart Cathleen A. , Ohlendorf Douglas H. , Schlievert Patrick M. , Dinges Martin M. , Cook William J.
Published: February 2009

The structure of toxic shock syndrome toxin‐1 (TSST‐1), the causative agent in toxic shock syndrome, has been determined in three crystal forms. The three structural models have been refined to R‐factors of 0.154, 0.150, and 0.198 at resolutions of 2.05 Å, 2.90 Å, and 2.75 Å, respectively. One crystal form of TSST‐1 contains a zinc ion bound between two symmetry‐related molecules. Although not required for biological activity, zinc dramatically potentiates the mito‐genicity of TSST‐1 at very low concentrations. In addition, the structure of the tetramutant TSST‐1H [T691, Y80W, E132K, I140T], which is nonmitogenic and does not amplify endotoxin shock, has been determined and refined in a fourth crystal form (R‐factor = 0.173 to 1.9 Å resolution).
On the calculation of binding free energies using continuum methods: Application to MHC class I protein‐peptide interactions
Authors: Froloff Nicolas , Windemuth Andreas , Honig Barry
Published: February 2009

This paper describes a methodology to calculate the binding free energy (ΔG) of a protein‐ligand complex using a continuum model of the solvent. A formal thermodynamic cycle is used to decompose the binding free energy into electrostatic and non‐electrostatic contributions. In this cycle, the reactants are discharged in water, associated as purely nonpolar entities, and the final complex is then recharged. The total electrostatic free energies of the protein, the ligand, and the complex in water are calculated with the finite difference Poisson‐Boltzmann (FDPB) method. The nonpolar (hydrophobic) binding free energy is calculated using a free energy‐surface area relationship, with a single alkane/water surface tension coefficient (γaw). The loss in backbone and side‐chain configurational entropy upon binding is estimated and added to the electrostatic and the nonpolar components of ΔG. The methodology is applied to the binding of the murine MHC class I protein H‐2Kb with three distinct peptides, and to the human MHC class I protein HLA‐A2 in complex with five different peptides. Despite significant differences in the amino acid sequences of the different peptides, the experimental binding free energy differences (ΔΔGexp) are quite small (<0.3 and <2.7 kcal/mol for the H‐2Kb and HLA‐A2 complexes, respectively). For each protein, the calculations are successful in reproducing a fairly small range of values for ΔΔGcalc (<4.4 and <5.2 kcal/mol, respectively) although the relative peptide binding affinities of H‐2Kb and HLA‐A2 are not reproduced. For all protein‐peptide complexes that were treated, it was found that electrostatic interactions oppose binding whereas nonpolar interactions drive complex formation. The two types of interactions appear to be correlated in that larger nonpolar contributions to binding are generally opposed by increased electrostatic contributions favoring dissociation. The factors that drive the binding of peptides to MHC proteins are discussed in light of our results.
Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR
Authors: Chung Evonne W. , Nettleton Ewan J. , Morgan Charles J. , Groß Michael , Miranker Andrew , Radford Sheena E. , Dobson Christopher M. , Robinson Carol V.
Published: February 2009

The extent of deuterium labeling of hen lysozyme, its three‐disulfide derivative, and the homologous α‐lactalbumins, has been measured by both mass spectrometry and NMR. Different conformational states of the proteins were produced by varying the solution conditions. Alternate protein conformers were found to contain different numbers of 2H atoms. Furthermore, measurement in the gas phase of the mass spectrometer or directly in solution by NMR gave consistent results. The unique ability of mass spectrometry to distinguish distributions of 2H atoms in protein molecules is exemplified using samples prepared to contain different populations of 2H‐labeled protein. A comparison of the peak widths of bovine α‐lactalbumin in alternate solution conformations but containing the same average number of 2H atoms showed dramatic differences due to different 2H distributions in the two protein conformers. Measurement of 2H distributions by ESI‐MS enabled characterization of conformational averaging and structural heterogeneity. In addition, a time course for hydrogen exchange was examined and the variation in distributions of 2H atom compared with simulations for different hydrogen exchange models. The results clearly show that exchange from the native state of bovine α‐lactalbumin at 15°C is dominated by local unfolding events.
The adaptability of Escherichia coli thioredoxin to non‐conservative amino acid substitutions
Authors: O'Brien R. , Driscoll P. C. , Davis B. , Ladbury J. E. , Wynn R. , Plaxco K. W. , Sturtevant J. M.
Published: February 2009

The adaptability of Escherichia coli thioredoxin to the substitution of a series of non‐natural amino acids has been investigated. Different thiosulfonated alkyl groups were inserted into the hydrophobic core of the protein in position 78 via disulfide bonding with a buried cysteine residue as previously described (Wynn R, Richards FM. 1993. Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. Protein Sci 2:395‐403). The side chains added to the cysteine included methyl, ethyl, n‐propyl, n‐butyl, n‐pentyl, and cyclo‐pentyl derivatives. The side chains appear to exploit the presence of the large cavities to incorporate these variant forms, enabling the protein to fold and have some activity. Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein. Thermodynamic data revealed that the entropic effect of restricting the side chains in the folded protein has an effect on the stability. The variant forms of thioredoxin have different propensities to form dimers despite the limited structural perturbations. Molecular modeling studies allow the conformation of the side chains to be assessed.
How protein chemists learned about the hydrophobic factor
Authors: Tanford Charles
Published: February 2009

It is generally accepted today that the hydrophobic force is the dominant energetic factor that leads to the folding of polypeptide chains into compact globular entities. This principle was first explicitly introduced to protein chemists in 1938 by Irving Langmuir, past master in the application of hydrophobicity to other problems, and was enthusiastically endorsed by J.D. Bernal. But both proposal and endorsement came in the course of a debate about a quite different structural principle, the so‐called “cyclol hypothesis” proposed by D. Wrinch, which soon proved to be theoretically and experimentally unsupportable. Being a more tangible idea, directly expressed in structural terms, the cyclol hypothesis received more attention than the hydrophobic principle and the latter never actually entered the mainstream of protein science until 1959, when it was thrust into the limelight in a lucid review by W. Kauzmann. A theoretical paper by H.S. Frank and M. Evans, not itself related to protein folding, probably played a major role in the acceptance of the hydrophobicity concept by protein chemists because it provided a crude but tangible picture of the origin of hydrophobicity per se in terms of water structure.
Hydrophobic folding units at protein‐protein interfaces: Implications to protein folding and to protein‐protein association
Authors: Tsai Chung‐Jung , Nussinov Ruth
Published: February 2009

A hydrophobic folding unit cutting algorithm, originally developed for dissecting single‐chain proteins, has been applied to a dataset of dissimilar two‐chain protein‐protein interfaces. Rather than consider each individual chain separately, the two‐chain complex has been treated as a single chain. The two‐chain parsing results presented in this work show hydrophobicity to be a critical attribute of two‐state versus three‐state protein‐protein complexes. The hydrophobic folding units at the interfaces of two‐state complexes suggest that the cooperative nature of the two‐chain protein folding is the outcome of the hydrophobic effect, similar to its being the driving force in a single‐chain folding. In analogy to the protein‐folding process, the two‐chain, two‐state model complex may correspond to the formation of compact, hydrophobic nuclei. On the other hand, the three‐state model complex involves binding of already folded monomers, similar to the association of the hydrophobic folding units within a single chain. The similarity between folding entities in protein cores and in two‐state protein‐protein interfaces, despite the absence of some chain connectivities in the latter, indicates that chain linkage does not necessarily affect the native conformation. This further substantiates the notion that tertiary, non‐local interactions play a critical role in protein folding. These compact, hydrophobic, two‐chain folding units, derived from structurally dissimilar protein‐protein interfaces, provide a rich set of data useful in investigations of the role played by chain connectivity and by tertiary interactions in studies of binding and of folding. Since they are composed of non‐contiguous pieces of protein backbones, they may also aid in defining folding nuclei.
Statistical significance of hierarchical multi‐body potentials based on Delaunay tessellation and their application in sequence‐structure alignment
Authors: Munson Peter J. , Singh Raj K.
Published: February 2009

Statistical potentials based on pairwise interactions between Cα atoms are commonly used in protein threading/fold‐recognition attempts. Inclusion of higher order interaction is a possible means of improving the specificity of these potentials. Delaunay tessellation of the Cα‐atom representation of protein structure has been suggested as a means of defining multi‐body interactions.

A large number of parameters are required to define all four‐body interactions of 20 amino acid types (204 = 160,000). Assuming that residue order within a four‐body contact is irrelevant reduces this to a manageable 8,855 parameters, using a nonredundant dataset of 608 protein structures.

Three lines of evidence support the significance and utility of the four‐body potential for sequence‐structure matching. First, compared to the four‐body model, all lower‐order interaction models (three‐body, two‐body, one‐body) are found statistically inadequate to explain the frequency distribution of residue contacts.

Second, coherent patterns of interaction are seen in a graphic presentation of the four‐body potential. Many patterns have plausible biophysical explanations and are consistent across sets of residues sharing certain properties (e.g., size, hydrophobicity, or charge).

Third, the utility of the multi‐body potential is tested on a test set of 12 same‐length pairs of proteins of known structure for two protocols: Sequence‐recognizes‐structure, where a query sequence is threaded (without gap) through the native and a non‐native structure; and structure‐recognizes‐sequence, where a query structure is threaded by its native and another non‐native sequence. Using cross‐validated training, protein sequences correctly recognized their native structure in all 24 cases. Conversely, structures recognized the native sequence in 23 of 24 cases. Further, the score differences between correct and decoy structures increased significantly using the three‐ or four‐body potential compared to potentials of lower order.
Structural determinants of specificity in the cysteine protease cruzain
Authors: Gillmor Sarah A. , Craik Charles S. , Fletterick Robert J
Published: February 2009

The structure of cruzain, an essential protease from the parasite Trypanosoma cruzi, was determined by X‐ray crystallography bound to two different covalent inhibitors. The cruzain S2 specificity pocket is able to productively bind both arginine and phenylalanine residues. The structures of cruzain bound to benzoyl‐Tyr‐Ala‐fluoromethyl ketone and benzoyl‐Tyr‐Ala‐fluoromethyl ketone at 2.2 and 2.1 Å, respectively, show a pH‐dependent specificity switch. Glu 205 adjusts to restructure the S2 specificity pocket, conferring tight binding to both hydrophobic and basic residues. Kinetic analysis of activated peptide substrates shows that substrates placing hydrophobic residues in the specificity pocket are cleaved at a broader pH range than hydrophilic substrates. These results demonstrate how cruzain binds both basic and hydrophobic residues and could be important for in vivo regulation of cruzain activity.
The X‐ray structure of a mutant eye lens βB2‐crystallin with truncated sequence extensions
Authors: Norledge B.V. , Slingsby C. , Trinkl S. , Jaenicke R.
Published: February 2009

β‐Crystallins are oligomeric eye lens proteins that are related to monomeric γ‐crystallins by domain swapping: like γ‐crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. βB2, a major component of β‐crystallin oligomers, self‐associates to a homodimer in solution. In two crystal structures of native βB2, the protein is a 222‐symmetric tetramer of eight domains. It has previously been shown that a mutant of rat βB2‐crystallin, in which the bulk of the N‐ and C‐terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 Å resolution X‐ray structure of the tetramer, βB2ΔNCI. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N‐ and C‐terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C‐terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non‐covalent) linker, as it does in native βB2. There is a knock‐on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20° rotation between sets of paired domains.
Comparison of straight chain and cyclic unnatural amino acids embedded in the core of staphylococcal nuclease
Authors: Richards Frederic M. , Wynn Richard , Harkins Paul C. , Fox Robert O.
Published: February 2009

We have determined by X‐ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this behavior and have suggested that it may arise from the addition of side‐chain vibrational and oscillational motions with each bond as a side chain grows away from the relatively rigid protein main chain and/or the population of multiple rotamers (Wynn R, Harkins P, Richards FM, Fox RO. 1996. Mobile unnatural amino acid side chains in the core of staphylococcal nuclease. Protein Sci 5:1026‐1031). Reduction of the number of degrees of freedom by cyclization of a side chain would be expected to constrain these motions. These side chains are in fact well defined in the structures described here. Over‐packing of the protein core results in a 1.O Å shift of helix 1 away from the site of mutation. Additionally, we have determined the structure of a side chain containing a single hydrogen to fluorine atom replacement on a methyl group. A fluorine atom is intermediate in size between methyl group and a hydrogen atom. The fluorine atom is observed in a single position indicating it does not rotate like methyl hydrogen atoms. This change also causes subtle differences in the packing interactions.
Bayesian statistical analysis of protein side‐chain rotamer preferences
Authors: Dunbrack Roland L. , Cohen Fred E.
Published: February 2009

We present a Bayesian statistical analysis of the conformations of side chains in proteins from the Protein Data Bank. This is an extension of the backbone‐dependent rotamer library, and includes rotamer populations and average x angles for a full range of ø,ψ values. The Bayesian analysis used here provides a rigorous statistical method for taking account of varying amounts of data. Bayesian statistics requires the assumption of a prior distribution for parameters over their range of possible values. This prior distribution can be derived from previous data or from pooling some of the present data. The prior distribution is combined with the data to form the posterior distribution, which is a compromise between the prior distribution and the data. For the X2, X3, and X4 rotamer prior distributions, we assume that the probability of each rotamer type is dependent only on the previous X rotamer in the chain. For the backbone‐dependence of the x1 rotamers, we derive prior distributions from the product of the ø‐dependent and ψ‐dependent probabilities. Molecular mechanics calculations with the CHARMM22 potential show a strong similarity with the experimental distributions, indicating that proteins attain their lowest energy rotamers with respect to local backbone‐side‐chain interactions. The new library is suitable for use in homology modeling, protein folding simulations, and the refinement of X‐ray and NMR structures.
The role of PII conformations in the calculation of peptide fractional helix content
Authors: Park Soon‐Ho , Shalongo William , Stellwagen Earle
Published: February 2009

Changes in the temperature, pH, ionic strength, or denaturant concentration of aqueous solutions of the monomeric non‐α‐helical peptide acety1YEAAAKEAPAKEAAAKAamide generate changes in its dichroic spectrum characteristic for a conformational transition. This transition has the characteristic features of a residue PII/unstructured conformational equilibrium in which PII denotes an extended left‐handed helical conformation and unstructured denotes all the remaining conformations in a random coil ensemble. Replacement of the proline residue facilitates population of residues in an α‐helical conformation. However, the elliptcity values for these non‐proline peptides merge with the ellipticity of the proline peptide as the population of residues in the α‐helix conformation is diminished. This convergence suggests that all residues in a host/guest peptide series of the same length share a common PII/unstructured conformational equilibrium in a given solvent. We propose that the fractional helix content of peptides within such a series may be estimated by using a two‐state calculation in which the ellipticity for the non‐α‐helix conformations is provided by a peptide having a central proline guest residue.
Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: Structure and stability of the recombinant enzyme and its isolated cellulose‐binding domain
Authors: Wassenberg Doris , Schurig Hartmut , Jaenicke Rainer , Liebl Wolfgang
Published: February 2009

The hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120‐kDa endo‐l,4‐D‐xylanase exhibiting high intrinsic stability and a temperature optimum —90 °C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C‐terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose‐binding domain within the multi‐domain 120 kDa Xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431‐444).

In order to characterize the domain organization and the stability of XynA and its C‐terminal cellulose‐binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand‐free form, was expressed as a glutathione S‐transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield >80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far‐UV circular dichroism in order to characterize protein stability, guanidine‐induced unfolding of XynA leads to biphasic transitions, with half‐concentrations c1/2(GdmC1) ∼4 M and >5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine‐transition from 5 to 7 M GdmC1. This can be tentatively attributed to the cellulose‐binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi‐state behavior of XynA. In the case of CBD, a temperature‐induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far‐UV CD in the native state.
An unexpected structural relationship between integral membrane phosphatases and soluble haloperoxidases
Authors: Neuwald Andrew F.
Published: February 2009

The mechanism of a membrane‐bound enzyme important in phospholipid signaling, type 2 phosphatidic acid phosphatase, is suggested by sequence motifs shared with a soluble vanadium‐dependent chloroperoxidase of known structure. These regions are also conserved in other soluble globular and membrane‐associated proteins, including bacterial acid phosphatases, mammalian glucose‐6‐phosphatases, and the Drosophila developmental protein Wunen. This implies that a similar arrangement of catalytic residues specifies the active site within both soluble and membrane spanning domains.
CD6 recognizes the neural adhesion molecule BEN
Authors: Skonier John E. , Bowen Michael A. , Aruffo Alejandro , Bajorath JÜRgen
Published: February 2009

CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) have been detected on various immune cells and in the brain. CD6‐ligand interactions have been implicated in the regulation of T cell function. ALCAM shares the same extracellular domain organization and significant sequence homology with the chicken neural adhesion molecule BEN. Although ALCAM's CD6 binding site is only partially conserved in BEN, CD6 specifically binds BEN, albeit with ˜ 10‐fold lower avidity than ALCAM. Differences in binding avidity are not detected when ALCAM and BEN fusion proteins containing the full‐length extracellular regions are tested. Homotypic interactions between full‐length forms are likely to account for these observations. The identified cross‐species interaction between CD6 and BEN suggests that CD6‐ligand interactions are highly conserved.
Crystallization of 5‐aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X‐ray characterization of the crystals
Authors: Erskine P.T. , Cooper J. , Lambert R. , Lewis G. , Wood S.P. , Shoolingin‐Jordan P.M. , Maignan S. , Spencer P. , Senior N. , Awan S. , Warren M. , Tickle I.J. , Thomas P.
Published: February 2009

5‐Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5‐aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo‐octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group 1422 (unit cell dimensions a = b= 130.7 Å, c = 142.4 Å). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (1422) but with a smaller unit cell volume (a = b = 103.7 Å, c = 167.7 Å). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.
Surface topology of Minibody by selective chemical modifications and mass spectrometry
Authors: Zappacosta Francesca , Ingallinella Paolo , Scaloni Andrea , Pessi Antonello , Bianchi Elisabetta , Sollazzo Maurizio , Tramontano Anna , Marino Gennaro , Pucci Piero
Published: February 2009

The surface topology of the Minibody, a small de novo‐designed β‐protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values.

The results of this study indicate that, while overall the Minibody model is correct, the β‐sheet formed by the N‐ and C‐terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini s, Sollazzo M, Tramontano A, Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5902‐813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X‐ray structure cannot be obtained due to insufficient solubility of the molecule.
Identification of the epitopes of calcitonin gene‐related peptide (CGRP) for two anti‐CGRP monoclonal antibodies by 2D NMR
Authors: Hubbard JULIA A.M. , Raleigh Daniel P. , Bonnerjea Julian R. , Dobson Christopher M.
Published: February 2009

The interactions between calcitonin gene‐related peptide and FAB fragments prepared from two different high‐affinity anti‐CGRP monoclonal antibodies (CB3 and CD1) have been studied at physiological pH using the ability of 1H NMR to detect selectively regions of dynamic flexibility. The 37‐residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However, the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C‐terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CD1, the conformationally restricted residues appear to lie predominantly within the N‐terminal half of the CGRP molecule, particularly residues 4‐16, although several residues in the middle section of the sequence (22‐31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.
Empirical free energy calculation: Comparison to calorimetric data
Authors: Weng Zhiping , Delisi Charles , Vajda Sandor
Published: February 2009

An effective free energy potential, developed originally for binding free energy calculation, is compared to calorimetric data on protein unfolding, described by a linear combination of changes in polar and nonpolar surface areas. The potential consists of a molecular mechanics energy term calculated for a reference medium (vapor or nonpolar liquid), and empirical terms representing solvation and entropic effects. It is shown that, under suitable conditions, the free energy function agrees well with the calorimetric expression. An additional result of the comparison is an independent estimate of the side‐chain entropy loss, which is shown to agree with a structure‐based entropy scale. These findings confirm that simple functions can be used to estimate the free energy change in complex systems, and that a binding free energy evaluation model can describe the thermodynamics of protein unfolding correctly. Furthermore, it is shown that folding and binding leave the sum of solute‐solute and solute‐solvent van der Waals interactions nearly invariant and, due to this invariance, it may be advantageous to use a nonpolar liquid rather than vacuum as the reference medium.
The effect of Arg306 → Ala and Arg506 → Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S
Authors: Egan Jack O. , Kalafatis Michael , Mann Kenneth G.
Published: February 2009

Factor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site‐directed mutagenesis of human factor V cDNA was used to substitute Arg306 → Ala (rfVa306A) and Arg506 → Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by α‐thrombin and their inactivation by APC were assessed in coagulation assays using factor V‐deficient plasma. All recombinant and wild‐type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild‐type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90‐min incubation with APC, rfVa306A and rfva506Q retain approximately 30‐40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90‐min incubation in the presence of APC. Recombinant maWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg30h. Over‐all, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.
Calcium binding to tandem repeats of EGF‐like modules. Expression and characterization of the EGF‐like modules of human Notch‐1 implicated in receptor‐ligand interactions
Authors: Rand Matthew D. , Lindblom Anders , Carlson Joyce , Villoutreix Bruno O. , Stenflo Johan
Published: February 2009

The Ca2+‐binding epidermal growth factor (cbEGF)‐like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch‐1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca2+‐binding. We report the Ca2+‐binding properties of EGF11‐12 and EGF10‐13 from human Notch‐1 (hNEGF11‐12 and hNEGF10‐13), modules previously shown to support Ca2+‐dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5′‐Br2BAPTA and 5‐NBAPTA, gave two binding constants for hNEGF11‐12, Kd1 = 3.4 × 10−5 M and Kd2 2.5 × 10−4 M. The high‐affinity site was found to be localized to hNEGF12. Titration of hNEGF10‐13 gave three binding constants, Kd1 = 3.1 × 10−6 M, Kd2 = 1.6 × 10−4 M, and Kd3 > 2.5 × 10−4 M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11‐12 and hNEGF10‐13 had 10 to 100‐fold higher affinity than reported for EGF32‐33 and EGF25‐31, respectively, from fibrillin‐1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11‐12 based on fibrillin‐1 EGF32‐33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca2+‐binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.
Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: A comparison with NAD bound to the oxidoreductase enzymes
Authors: Bell Charles E. , Yeates Todd O. , Eisenberg David
Published: February 2009

The conformation of NAD bound to diphtheria toxin (DT), an ADP‐ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)‐binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N‐glycosidic bond between the nicotinamide ring and the N‐ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase‐bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT's function as an ADP‐ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle XN ∼ 0° about the scissile N‐glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle XN ∼ 0° could contribute to catalysis, possibly by orienting the C1′N atom of NAD for nucleophilic attack, or by placing strain on the N‐glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)‐binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel β‐sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C‐terminal end of a parallel β‐sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP‐ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP‐ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.