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At least 217 records · Page 12Linked to original sources
PubMed1988

Structure generation by reduction: a new strategy for computer-assisted structure elucidation.

A problem common to computer programs for structure elucidation is the efficient and prospective use of the input information to constrain the structure generation process. The input may consist of potentially overlapping substructure requirements and alternative substructure interpretations of spectral data. Other useful information may be structural features that must not be present in the output structures. All of these may interact in a complex manner that is impossible to determine by use of a bond-by-bond structure assembly algorithm. A new method is described called structure reduction. In contrast to structure assembly, this method begins with a set of all bonds and removes inconsistent bonds as structure generation progresses. This results in a more efficient use of the input information and the ability to use potentially overlapping required substructures. Several examples illustrate the application of our computer program COCOA, which uses this method to solve real-world structure elucidation problems.

Chemical Phenomena↗
PubMed2003

Structure and bonding of Sr3In11: how size and electronic effects determine structural stability of polar intermetallic compounds.

The binary compound Sr(3)In(11) (SrIn(3.667)) was synthesized and structurally characterized by X-ray diffraction experiments. It crystallizes in the orthorhombic La(3)Al(11) structure type (space group Immm, Z = 2; a = 4.9257(6), b = 14.247(2), c = 11.212(2) A). The crystal structure of Sr(3)In(11) bears features of the monoclinic EuIn(4) structure, which is adopted by SrIn(4), and the prominent tetragonal BaAl(4) structure. Sr(3)In(11) is stable until 550 degrees C. At higher temperatures it decomposes peritectically into SrIn(2) and In. Structural stability and bonding properties of Sr(3)In(11) were investigated by first principles calculations and compared to SrIn(4) in the monoclinic EuIn(4) and the tetragonal BaAl(4) structure. All three structures consist of a three-dimensional, polyanionic, network formed by In atoms and Sr cations encapsulated in cages. For the BaAl(4)-type SrIn(4), In-In network bonding is perfectly optimized. In contrast, the networks of EuIn(4)-type SrIn(4) and Sr(3)In(11) appear hypo- and hyperelectronic, respectively. The formation of Sr(3)In(11) with a composition close to 1:4 and the nonexistence of BaAl(4)-type SrIn(4) is explained by a delicate interplay of size and electronic factors governing structural stability in the In-rich part of the Sr-In system.

Journal Article↗
PubMed1996

The Four-Connected Net in the CeCu(2) Structure and Its Ternary Derivatives. Its Electronic and Structural Properties.

The crystallochemistry of and the bonding in the orthorhombic four-connected nets of BaIn(2) (CeCu(2) structure) and of CaPtSn (TiNiSi structure, a derivative of the CeCu(2) structure) are analyzed with approximate molecular orbital calculations. Following the Zintl concept, in BaIn(2) the In(-) ions are isoelectronic with group IV tin and should adopt a four-connected structure. In contrast to alpha-tin, which has a cubic diamond structure, the indium ions in BaIn(2) build up an orthorhombic three-dimensional four-connected net containing distorted tetrahedra and ladder polymers of four-membered rings. In the CeCu(2) structure (space group Imma) two bond angles in these distorted tetrahedra are fixed at 90 degrees. The four-connected net in the CeCu(2) structure is topologically related to the layers in black phosphorus (space group Cmca). In CaPtSn (TiNiSi structure) the orthorhombic four-connected net is formed by (PtSn)(2)(-) ions in an ordered arrangement. Calculations on BaIn(2) and CaPtSn show that the four-connected nets are increasingly stabilized as the valence electron count is increased from 16 to 30 valence electrons per 4 formula units. For more than 30e, the nets are destabilized due to filling of M-E antibonding states. Structural data obtained by precise single crystal investigations for the TiNiSi series CaPdIn (20e), CaPdSn (24e), CaPdSb (28e), and CaAgSb (32e), confirm the results of the extended Hückel calculations. We find an interesting and understandable angular asymmetry of the tetrahedral sites in these ternary compounds.

Journal Article↗
PubMed1999

The three-dimensional structure of Asp189Ser trypsin provides evidence for an inherent structural plasticity of the protease.

Trypsin mutant Asp189Ser, first described by Gráf et al. [Gráf, L., Jancsó, A., Szilágyi, L., Hegyi, G., Pintér, K., Náray-Szabó, G., Hepp, J., Medzihradszky, K. & Rutter, W.J. (1988) Proc. Natl Acad. Sci. USA 85, 4961-4965] has played an important role in recent studies on the structural basis of substrate-specific catalysis by serine proteases. The present work reports the three-dimensional structure of this mutant crystallized in unliganded form: the first unliganded rat trypsin structure reported. The X-ray structure of the Asp189Ser trypsin mutant in complex with bovine pancreatic trypsin inhibitor is already known. The X-ray structure of free Asp189Ser rat trypsin revealed that the single amino acid mutation at the bottom of the substrate binding pocket of trypsin resulted in extensive structural changes around the mutated site and in dimerization of the mutant, in contrast with the complexed enzyme the structure of which is practically the same as that of wild-type trypsin. The structural rearrangement in the mutant was shown to be restricted to the activation domain region providing further evidence for the allosteric property of this structural-functional unit of the enzyme. This study supports our view that the plasticity of the activation domain may play an important role in the mechanism of substrate-specific serine protease action.

Allosteric Site↗
PubMed2004

Theoretical prediction of electronic structures of fully pi-conjugated zinc oligoporphyrins with curved surface structures.

A theoretical prediction of the electronic structures of fully pi-conjugated zinc oligoporphyrins with curved surface, ring, tube, and ball-shaped structures was conducted as the objective for the future development of triply meso-meso-, beta-beta-, and beta-beta-linked planar zinc oligoporphyrins. The excitation energies and oscillator strengths for the optimal ring and ball structures were calculated using the time-dependent density functional theory (DFT). Although there is an extremely small energy difference of < 0.1 eV between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the ring structure relative to the same-sized triply linked planar one, the Q and B bands of the former are smaller redshifted excitation energies and intensified oscillator strengths than those of the latter due to the structurally shortened effective pi-conjugated lengths for the electron transition. It is expected that the ball structure becomes an excellent electron acceptor and shows the highly redshifted Q' band in the near-IR region relative to the monomer. The minimum value of the HOMO-LUMO energy gaps of the infinite-length ring structures was estimated using periodic boundary conditions within the DFT, resulting in the metallic characters of both the tube structures with and without the spiral triply linked porphyrin array. The relation between the diameters and strain energies of the tube and ball structures was also examined. The present fused zinc porphyrins may become more colorful materials with new optelectronic properties including artificial photosynthesis than the carbon nanotubes and fullerenes when the axial coordinations of the central metal of porphyrins are functionally used.

Journal Article↗
PubMed1998

On the structural changes of native human alpha2-macroglobulin upon proteinase entrapment. Three-dimensional structure of the half-transformed molecule.

The reconstructions of an intermediate form of human alpha2-macroglobulin (half-transformed alpha2M) in which two of its four bait regions and thiol ester sites were cleaved by chymotrypsin bound to Sepharose were obtained by three-dimensional electron microscopy from stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a structure with approximate dimensions of 195 (length) x 135 (width) and 130 A (depth) with an internal funnel-shaped cavity. The structure shows that a chisel-shaped body is connected to a broad base at the opposing end by four stands. Four approximately 45 A diameter large openings in the body of the structure result in a central cavity that is more accessible to the proteinase than those associated with the native or fully transformed structures. The dissimilarity in the shapes between the two ends of alpha2M half-transformed and the similarity between its chisel-shaped body and that of native alpha2M indicate that the chymotrypsin has cleaved both bait regions in the bottom-half of the structure. Consequently, its functional division lies on the minor axis. The structural organization is in accord with biochemical studies, which show that the half-transformed alpha2M migrates on native polyacrylamide gels at a rate intermediate to the native and fully transformed alpha2M and is capable of trapping 1 mol of proteinase. Even though its upper portion is similar to the native molecule, significant differences in their shapes are apparent and these differences may be related to its slower reaction with a proteinase than the native structure. These structural comparisons further support the view that the transformation of alpha2M involves an untwisting of its strands with an opening of the cavity for entrance of the proteinase and a retwisting of the strands around the proteinase resulting in its encapsulation.

Binding Sites↗
PubMed1990

Molecular-mechanics modelling of drug-DNA structures; the effects of differing dielectric treatment on helix parameters and comparison with a fully solvated structural model.

This study analyses the influence that the nature of the dielectric constant has on the final structures obtained from in vacuo molecular mechanics calculations on a drug-DNA complex and compares these structures with the energy minimised complex including explicit solvent molecules. Minimisations have been performed on a proflavine-decanucleotide structure, where the drug was intercalated at the d(CpG) site of the d(GpApTpApCpGpApTpApC) decamer duplex, using two expressions for the dielectric constant: a distance-independent, epsilon ij = EPS, and a distance-dependent, epsilon ij = EPS*Rij, form and for values of EPS from 1 to 8. Significantly different structures are obtained for the distance-independent and the distance-dependent expressions of the dielectric constant. The use of a distance-independent dielectric constant leads to distorted structures, which are very sensitive to slight changes in the value of EPS. The use of a distance-dependent dielectric constant leads to less distorted and more stable structures. The effects on helical parameters are analysed in detail. The structures obtained for different values of EPS (within the distance-dependent formalism) seem to converge for values of EPS equal to 4 or greater. Based on these results a distance-dependent form of the dielectric with an EPS value of 4 is recommended in order to produce reliable refined nucleic acid structures by molecular mechanics. These conclusions have been supported by molecular-mechanics minimisation of the same structure with the inclusion of explicit water molecules and counter-ions.

Base Composition↗
PubMed1996

The FSSP database: fold classification based on structure-structure alignment of proteins.

The FSSP database presents a continuously updated classification of 3-D protein folds based on an all-against-all comparison of structures currently in the Protein Data Bank (PDB) [Bernstein et al. (1977) J. Mol. Biol., 112, 535- 542]. The database currently contains an extended structural family for each of 600 representative protein chains which have <25% mutual sequence identity. The results of the exhaustive pairwise structure comparisons are reported in the form of a fold tree generated by hierarchical clustering and as a series of structurally representative sets of folds at varying levels of uniqueness. For each query structure from the representative set, there is a database entry containing structure-structure alignments with its structural neighbours in the representative set and its sequence homologs in the PDB. All alignments are based purely on the 3-D co-ordinates of the proteins and are derived by an automatic structure comparison program (Dali). The FSSP database is accessible electronically on the World Wide Web and by anonymous ftp.

Amino Acid Sequence↗
PubMed2002

The structure of orange HgI2. II. Diamond-type structure and twinning.

The metastable orange crystals of HgI(2) comprise three different crystal structures all of which are built from corner-linked Hg(4)I(10) supertetrahedra. Two of the structures are end members with the maximum degree of order (MDO) of a polytypic layer structure. In this paper, the third structure (D) determined from X-ray diffraction, a crystal chemical discussion of the four known tetrahedral HgI(2) structures, and a twinning model are presented. All the various diffraction results published during the past 70 years are now explained. The Hg(4)I(10) supertetrahedra of the tetragonal structure D are corner-linked into two interpenetrating diamond-type networks. The stable red form and the three orange structures show the same cubic densest packing of I atoms and differ only in the distribution of Hg atoms in the tetrahedral voids. Transformations between the structures may involve only movements of Hg atoms, as implied by larger thermal displacement parameters of Hg than of I. A multiply twinned conglomerate of MDO1, MDO2 and D, each structure occurring in three orientations, results in metrically cubic crystals whose Bragg reflections are very close to reciprocal lattice points.

Journal Article↗
PubMed1999

Structure and function of the N-linked glycans of HBP/CAP37/azurocidin: crystal structure determination and biological characterization of nonglycosylated HBP.

The three N-glycosylation sites of human heparin binding protein (HBP) have been mutated to produce a nonglycosylated HBP (ng-HBP) mutant. ng-HBP has been crystallized and tested for biological activity. Complete X-ray data have been collected to 2.1 A resolution, and the structure has been fully refined to an R-factor of 18.4% (R(free) 27.7%). The ng-HBP structure reveals that neither the secondary nor tertiary structure have changed due to the removal of the glycosylation, as compared to the previously determined glycosylated HBP structure. Although the primary events in N-linked glycosylation occurs concomitant with polypeptide synthesis and therefore possesses the ability to influence early events in protein folding, we see no evidence of glycosylation influencing the structure of the protein. The root-mean-square deviation between the superimposed structures was 0.24 A (on C alpha atoms), and only minor local structural differences are observed. Also, the overall stability of the protein seems to be unaffected by glycosylation, as judged by the B-factors derived from the two X-ray structures. The flexibility of a glycan site may be determined by the local polypeptide sequence and structure rather than the glycan itself. The biological in vitro activity assay data show that ng-HBP, contrary to glycosylated HBP, mediates only a very limited stimulation of the lipopolysaccharide induced cytokine release from human monocytes. In animal models of fecal peritonitis, glycosylated HBP treatment rescues mice from and an otherwise lethal injury. It appears that ng-HBP have significant effect on survival, and it can be concluded that ng-HBP can stimulate the host defence machinery albeit to a lesser extent than glycosylated HBP.

Animals↗
PubMed2002

Smoothing of pixelated finite element models of cancellous bone structures and the effect on the predicted structural properties of the bone.

Many areas of biomedical engineering involve the modelling of biological systems, often using data from medical scanning techniques such as computed microtomography (microCT), and the prediction of the mechanical properties of these systems via finite element models. These models, and also those produced from remodelling simulations on idealized bone structures, are inherently highly pixelated and therefore have a high degree of surface roughness. The purpose of this paper is to demonstrate that this surface roughness need not necessarily have an influence on the predicted properties of the object under examination. To demonstrate this, two-dimensional idealized models of cancellous bone structures were used that were initially depleted and then rebuilt stochastically. A hysteresis effect was observed such that a significant amount of rebuilding beyond the original density was required to regain the initial intact stiffness. To ensure that this effect was not an artefact of the high degree of surface roughness of the rebuilt structures, a two-stage smoothing procedure was applied to assess if this had any effect on the stiffness of the structures. The superpixelation of the structures appeared to have a more profound effect than the smoothing procedures, although the smoothed structures still had stiffness and density values similar to those of the original structures, with a hysteresis effect still evident. This proves that the pixelization of the structures does not have a significant effect on the predicted mechanical properties of the structures. This work has important implications for other models that exhibit a high degree of surface roughness.

Artifacts↗
PubMed2000

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.

Blotting, Western↗
PubMed2000

[The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal. IV. Structural heterogeneity of stretched chromatin in membrane-free cell nuclei from human peripheral lymphocytes].

Densely aggregated chromatin of mature human or animal peripheral lymphocytes is inaccessible for structural investigation on preparations of both intact cell and conventionally spread chromatin. Giemsa- and DAPI-positive "free chromatin" structures, in addition to amembraneous nuclei, were isolated from intact lymphocytes gently treated with Triton-X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol-glacial acetic acid (3:1), revealed three main types of these "free chromatin" structures: dense chromatin structures (DCS), loose chromatin structures (LCS) and nuclear spreads (NS). The share of each nuclear derivative may be shifted by changing either detergent concentration and(or) the time of incubation in detergent solution. Each DSC consists of condensed "residual" nucleus, similar in from and size with an intact lymphocyte nucleus, and involves 1-15 uni- or olygonemic chromatin sprouts of different length. LSC contain heterogeneously loosened spindle-shape or drop-like nuclei, being several times longer and wider than DCS-nuclei, and 1-3 long uni- or olygonemic chromatin tail-pieces and incidentally observed lateral chromatin sprouts. The majority of LCS contain either a chromocenter of different number of end-to-end associated spindle-shape domains of condensed chromatin. The latter reached 2-5 x 1.5 microns being cross-striated or spiral in structure. NS represent spread chromatin fibrillar structures varying from 150 to 500 microns in length and from 1.5 to almost 50 microns in width. NS consist of 0.3-0.4 micron smooth and 0.4-0.8 micron beaded chromatin fibres. Thin fibres produce web-like domains of NS. and thick fibres form olygonemic bundles or end-to-end association of unit chromatin fibres within NS. Some portion of thick unit fibres of NS gave rise to local splitting into two thin fibres with a similar bead patterns. Thick argyrophilic fibers of the nucleolus also displayed a beaded structure and commonly spread hand-in-hand with the basic chromatin fibre aggregations.

Cell Nucleus↗
PubMed1994

Protein structural similarities predicted by a sequence-structure compatibility method.

A method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3-dimensional structures and identifies the most likely structure. The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH). The functional and evolutionary implications of the predictions are discussed. (1) The structural similarity between SKase and adenylate kinase was predicted. Alignment of their sequences reveals that the ATP-binding type A sequence motif and 2 ATP-binding arginine residues are conserved. The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship. (2) The structural similarity between IIABMan and galactose/glucose-binding protein (GGBP) was predicted. The IIA and IIB domains are aligned with the N- and C-terminal domains of GGBP, respectively. The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate-binding cleft of GGBP. The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate. (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase beta subunit. Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins.

Amino Acid Sequence↗
PubMed2001

Examination of possible structural constraints of MHC-binding peptides by assessment of their native structure within their source proteins.

Antigenic peptides bind to major histocompatibility complex (MHC) molecules as a prerequisite for their presentation to T cells. In this study, we investigate possible structural preferences of MHC-binding peptides by examining the conformation space defined by the structures of these peptides within their native source proteins. Comparison of the conformation space of the native structures of MHC-binding nonamers and a corresponding conformation space defined by a random set of nonamers showed no significant difference. This suggests that the environment of the MHC binding groove has evolved to bind peptides with essentially any "structural background." A slight tendency for an extended beta-conformation at positions 8 and 9 was observed for the set of native structures. We suggest that such a preference may facilitate the binding of the C-terminal anchor position of processed peptides into the corresponding specificity pocket. MHC-binding peptides represent examples of short subsequences that are present in two different structural environments: within their native protein and within the MHC binding groove. Comparison of the native and of the bound structure of the peptides showed that peptides up to 14 residues long may adopt different conformations within different protein environments. This has direct implications for structure prediction algorithms.

Amino Acid Sequence↗
PubMed1995

Solution structure of (Cd2+)1-calbindin D9k reveals details of the stepwise structural changes along the Apo-->(Ca2+)II1-->(Ca2+)I,II2 binding pathway.

The three-dimensional solution structure of (Cd2+)1-calbindin D9k has been determined by distance geometry, restrained molecular dynamics and relaxation matrix calculations using experimental constraints obtained from two-dimensional 1H and 15N-1H NMR spectroscopy. The final input data consisted of 1055 NOE distance constraints and 71 dihedral angle constraints, corresponding to 15 constraints per residue on average. The resulting ensemble of 24 structures has no distance or dihedral angle constraints consistently violated by more than 0.07 A and 1.8 degrees, respectively. The structure is characteristic of an EF-hand protein, with two helix-loop-helix calcium binding motifs joined by a flexible linker, and a short anti-parallel beta-type interaction between the two ion-binding sites. The four helices are well defined with a root mean square deviation from the mean coordinates of 0.35 A for the backbone atoms. The structure of the half-saturated cadmium state was compared with the previously determined solution structures of the apo and fully calcium saturated calbindin D9k. The comparisons were aided by introducing the ensemble averaged distance difference matrix as a tool for analyzing differences between two ensembles of structures. Detailed analyses of differences between the three states in backbone and side-chain dihedral angles, hydrogen bonds, interatomic distances, and packing of the hydrophobic core reveal the reorganization of the protein that occurs upon ion binding. Overall, it was found that (Cd2+)1-calbindin D9k, representing the half-saturated calcium state with an ion in site II, is structurally more similar to the fully calcium-saturated state than the apo state. Thus, for the binding sequence apo-->(Ca2+)II1-->(Ca2+)I,II2, the structural changes occurring upon ion binding are most pronounced for the first binding step, an observation that bears significantly on the molecular basis for cooperative calcium binding in calbindin D9k.

Animals↗
PubMed1997

The crystal structure of an Fe-superoxide dismutase from the hyperthermophile Aquifex pyrophilus at 1.9 A resolution: structural basis for thermostability.

Superoxide dismutase (SOD) from Aquifex pyrophilus, a hyperthermophilic bacterium, is an extremely heat-stable enzyme that maintains about 70% of its activity after heat treatment for 60 minutes at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we have determined the crystal structure of A. pyrophilus superoxide dismutase (Ap SOD), an Fe containing homotetrameric enzyme, at 1.9 A resolution, and compared it with SOD structures from a mesophile and a thermophile, and other enzyme structures from other hyperthermophiles. The structure has been refined to a crystallographic R-factor (I > 2sigma) of 17.0% and R-free (I > 2sigma) of 19.9%. While the overall structure of the Ap SOD monomer is similar to the other SODs, significant conformational differences are observed in a highly variable loop region and the C-terminal helix. The conformational differences in these regions alter the subunit arrangement of this enzyme and generate a very compact tetramer. Structural comparisons of three SODs have revealed that Ap SOD has some stabilizing features at both the tertiary and the quaternary structural level: The Ap SOD monomer contains a large number of ion-pairs and the Ap SOD tetramer has a dramatically increased buried surface area per monomer. Comparisons of the Ap SOD structure with that of other known enzymes from hyperthermophiles reveal that the increased number of intrasubunit ion-pairs is a common feature.

Amino Acid Sequence↗
PubMed1994

High resolution structures of the 4-4-20 Fab-fluorescein complex in two solvent systems: effects of solvent on structure and antigen-binding affinity.

Three-dimensional structures were determined for three crystal forms of the antigen binding fragment (Fab) of anti-fluorescein antibody 4-4-20 in complex with fluorescein. These included 1) a triclinic (P1) form crystallized in 47% (v/v) 2-methyl-2,4-pentanediol (MPD); 2) a triclinic (P1) form crystallized in 16% (w/v) poly(ethylene glycol), molecular weight 3350 (PEG); and 3) a monoclinic (P21) form crystallized in 16% PEG. Solvent molecules were added to the three models and the structures were refined to their diffraction limits (1.75-A, 1.78-A, and 2.49-A resolution for the MPD, triclinic PEG, and monoclinic PEG forms, respectively). Comparisons of these structures were interesting because 4-4-20 exhibited a lower antigen-binding affinity in 47% MPD (Ka = 1.3 x 10(8) M-1) than in either 16% PEG (Ka = 2.9 x 10(9) M-1) or phosphate-buffered saline (Ka = 1.8 x 10(10) M-1). Even though the solution behavior of the antibody was significantly different in MPD and PEG, the crystal structures were remarkably similar. In all three structures, the fluorescein-combining site was an aromatic slot formed by tyrosines L32, H96, and H97 and tryptophans L96 and H33. In addition, several active site constituents formed an electrostatic network with the ligand. These included a salt link between arginine L34 and one of fluorescein's enolate oxygen atoms, a hydrogen bond between histidine L27d and the second enolic group, a hydrogen bond between tyrosine L32 and the phenylcarboxylate group, and two medium range (approximately 5 A) electrostatic interactions with lysine L50 and arginine H52. The only major difference between the triclinic MPD and PEG structures was the degree of hydration of the antigen-combining site. Three water molecules participated in the above electrostatic network in the MPD structure, while eight were involved in the PEG structure. Based on this observation, we believe that 4-4-20 exhibits a lower affinity in MPD due to the depletion of the hydration shell of the antigen-combining site.

Amino Acid Sequence↗