bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractLanthanide 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

AbstractT‐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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractThis 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

AbstractThe 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

AbstractThe 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

AbstractIt 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

AbstractA 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

AbstractStatistical 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

AbstractThe 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

Abstractβ‐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.