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PubMed2003

Structural stability and solution structure of chaperonin GroES heptamer studied by synchrotron small-angle X-ray scattering.

The GroES protein from Escherichia coli is a well-known member of the molecular chaperones. GroES consists of seven identical 10 kDa subunits, and forms a dome-like oligomeric structure. In order to obtain information on the structural stability and unfolding-refolding mechanism of GroES protein, especially at protein concentrations (0.4-1.2 mM GroES monomer) that would mimic heat stress conditions in vivo, we have performed synchrotron small-angle X-ray scattering (SAXS) experiments. Surprisingly, in spite of the high protein concentration, reversibility in the unfolding-refolding reaction was confirmed by SAXS experiments structurally. Although the unfolding-refolding reaction showed an apparent single transition with a Cm of 1.1 M guanidium hydrochloride, a more detailed analysis of this transition demonstrated that the unfolding mechanism could be best explained by a sequential three-state model, which consists of native heptamer, dissociated monomer, and unfolded monomer. Together with our previous result that GroES unfolded completely via a partially folded monomer according to a three-state model at low protein concentration (5 microM monomer), the unfolding-refolding mechanism of GroES protein could be explained uniformly by the three-state model from low to high protein concentrations. Furthermore, to clarify an ambiguity of the native GroES structure in solution, especially mobile loop structures, we have estimated a solution structure of GroES using SAXS profiles obtained from experiments and simulation analysis. The result suggested that the native structure of GroES in solution was very similar to that seen in GroES-GroEL complex determined by crystallography.

Chaperonin 10↗
PubMed2001

Large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase from Thermococcus profundus studied by cryogenic X-ray crystal structure analysis and small-angle X-ray scattering.

Here we describe the large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase. Glutamate dehydrogenase from Thermococcus profundus is composed of six identical subunits of M(r) 46K, each with two distinct domains of roughly equal size separated by a large active-site cleft. The enzyme in the unligated state was crystallized so that one hexamer occupied a crystallographic asymmetric unit, and the crystal structure of the hexamer was solved and refined at a resolution of 2.25 A with a crystallographic R-factor of 0.190. In that structure, the six subunits displayed significant conformational variations with respect to the orientations of the two domains. The variation was most likely explained as a hinge-bending motion caused by small changes in the main chain torsion angle of the residue composing a loop connecting the two domains. Small-angle X-ray scattering profiles both at 293 and 338 K suggested that the apparent molecular size of the hexamer was slightly larger in solution than in the crystalline state. These results led us to the conclusion that (i) the spontaneous domain motion was the property of the enzyme in solution, (ii) the domain motion was trapped in the crystallization process through different modes of crystal contacts, and (iii) the magnitude of the motion in solution was greater than that observed in the crystal structure. The present cryogenic diffraction experiment enabled us to identify 1931 hydration water molecules around the hexamer. The hydration structures around the subunits exhibited significant changes in accord with the degree of the domain movement. In particular, the hydration water molecules in the active-site cleft were rearranged markedly through migrations between specific hydration sites in coupling strongly with the domain movement. We discussed the cooperative dynamics between the domain motion and the hydration structure changes in the active site of the enzyme. The present study provides the first example of a visualized hydration structure varying transiently with the dynamic movements of enzymes and may form a new concept of the dynamics of multidomain enzymes in solution.

Binding Sites↗
PubMed2003

Solution X-ray scattering data show structural differences among chimeras of yeast and chicken calmodulin: implications for structure and function.

We present here the first evidence, obtained by the use of small-angle X-ray scattering, of the solution structures of chimeras constructed from yeast (Saccharomyces cerevisiae, Sc) and chicken (Gallus gallus, Gg) calmodulin (CaM). The chimeric proteins used in this study are Sc(1-129)/Gg(130-148), Sc(1-128)/Gg(129-148), Sc(1-87)/Gg(88-148), and Sc(1-72)/Gg(73-148) CaMs, in which Sc(1-)(n)() and Gg(()(n)(+1)-148) descend from yeast and chicken CaM in the chimeric proteins, respectively. Under the Ca(2+)-saturated condition, the solution structure of Sc(1-128)/Gg(129-148) CaM has a dumbbell-like shape which is characteristic of vertebrate-type CaM, while that of Sc(1-129)/Gg(130-148) CaM takes an intermediate structure between the dumbbell-like shape and a compact globular shape. The results provide the direct evidence that the replacement of Asp(129) with Ser(129) induces an interaction between two lobes of Sc(1-129)/Gg(130-148) CaM and brings them close together. It implies that a site interacting with the N-lobe is induced in the C-lobe, although site IV that is unable to bind Ca(2+) hinders the ability of the C-lobe to undergo the conformational change to the full open state. In the presence of both Ca(2+) and a peptide synthesized to mimic the CaM binding domain on myosin light chain kinase, MLCK-22p, the solution structures of these chimeric CaMs take a similar compact globular shape but their interactions are quite different. The solution structure and interactions of Sc(1-72)/Gg(73-148) CaM are similar to those of Sc(1-87)/Gg(88-148) CaM. The structure of Sc(1-87)/Gg(88-148) CaM is similar to that of Sc(1-128)/Gg(129-148) CaM, but their interactions are different. The result indicates that the replacement of Glu(119) with Ala(119) has a critical effect on their interactions. Thus, the functional differences among these chimeric CaMs, which have been reported previously [Nakashima, K., et al. (1996) Biochemistry 35, 5602-5610], have been interpreted on the basis of the structures and interactions.

Amino Acid Sequence↗
PubMed2004

Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship.

Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.

Amino Acid Sequence↗
PubMed1999

Structural analysis of phospholipase A2 from functional perspective. 1. Functionally relevant solution structure and roles of the hydrogen-bonding network.

Bovine pancreatic phospholipase A2 (PLA2), a small (13.8 kDa) Ca2+-dependent lipolytic enzyme, is rich in functional and structural character. In an effort to examine its detailed structure-function relationship, we determined its solution structure by multidimensional nuclear magnetic resonance (NMR) spectroscopy at a functionally relevant pH. An ensemble of 20 structures generated has an average root-mean-square deviation (RMSD) of 0.62 +/- 0.08 A for backbone (N, Calpha, C) atoms and 0.98 +/- 0.09 A for all heavy atoms. The overall structure shows several notable differences from the crystal structure: the first three residues at the N-terminus, the calcium-binding loop (Y25-T36), and the surface loop (V63-N72) appear to be flexible; the alpha-helical conformation of helix B (E17-F22) is absent; helix D appears to be shorter (D59-V63 instead of D59-D66); and the hydrogen-bonding network is less defined. These differences were analyzed in relation to the function of PLA2. We then further examined the H-bonding network, because its functional role or even its existence in solution has been in dispute recently. Our results show that part of the H-bonding network (the portion away from N-terminus) clearly exists in solution, as evidenced by direct observation (at 11.1 ppm) of a strong H-bond between Y73 and D99 and an implicated interaction between D99 and H48. Analyses of a series of mutants indicated that the existence of the Y73.D99 H-bond correlates directly with the conformational stability of the mutant. Loss of this H-bond results in a loss of 2-3 kcal/mol in the conformational stability of PLA2. The unequivocal identification and demonstration of the structural importance of a specific hydrogen bond, and the magnitude of its contribution to conformational stability, are uncommon to the best of our knowledge. Our results also suggest that, while the D99.H48 catalytic diad is the key catalytic machinery of PLA2, it also helps to maintain conformational integrity.

Animals↗
PubMed1999

Solution structure of carnobacteriocin B2 and implications for structure-activity relationships among type IIa bacteriocins from lactic acid bacteria.

Carnobacteriocin B2 (CbnB2), a type IIa bacteriocin, is a 48 residue antimicrobial peptide from the lactic acid bacterium Carnobacterium pisicola LV17B. Type IIa bacteriocins have a conserved YGNGVXC sequence near the N-terminus and usually contain a disulfide bridge. CbnB2 seemed to be unique in that its two cysteines (Cys9 and Cys14) could be isolated as free thiols [Quadri et al. (1994) J. Biol. Chem. 26, 12204-12211]. To establish the structural consequences of the presence or absence of a disulfide bridge and to investigate if the YGNGVXC sequence is a receptor-binding motif [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429], the three-dimensional solution structure of CbnB2 was determined by two-dimensional (1)H nuclear magnetic resonance (NMR) techniques. Mass spectroscopic and thiol modification experiments on CbnB2 and on model peptides, in conjunction with activity measurements, were used to verify the redox status of CbnB2. The results show that CbnB2 readily forms a disulfide bond and that this peptide has full antimicrobial activity. NMR results indicate that CbnB2 in trifluoroethanol (TFE) has a well-defined central helical structure (residues 18-39) but a disordered N terminus. Comparison of the CbnB2 structure with the refined solution structure of leucocin A (LeuA), another type IIa bacteriocin, indicates that the central helical structure is conserved between the two peptides despite differences in sequence but that the N-terminal structure (a proposed receptor binding site) is not. This is unexpected because LeuA and CbnB2 exhibit >66% sequence identity in the first 24 residues. This suggests that the N-terminus, which had been proposed [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429] to be a receptor binding site of type IIa bacteriocins, may not be directly involved and that recognition of the amphiphilic helical portion is the critical feature.

Amino Acid Sequence↗
PubMed2006

Structure-based secondary structure-independent approach to design protein ligands: Application to the design of Kv1.2 potassium channel blockers.

We have developed a structure-based approach to the design of protein ligands. This approach is based on the transfer of a functional binding motif of amino acids, often referred as to the "hot spot", on a host protein able to reproduce the functional topology of these residues. The scaffolds were identified by a systematic in silico search in the Protein Data Bank for proteins possessing a group of residues in a topology similar to that adopted by the functional motif in a reference ligand of known 3D structure. In contrast to previously reported studies, this search is independent of the particular secondary structure supporting the functional motif. To take into account the global properties of the host protein, two additional criteria were taken into account in the selection process: (1) Only those scaffolds sterically compatible with the positioning of the functional motif as observed in a reference complex model were retained. (2) Host proteins displaying electrostatic potentials, in the region of the transferred functional motif, similar to that of the reference ligand were selected. This approach was applied to the development of protein ligands of the Kv1.2 channel using BgK, a small protein isolated from the sea anemone Bunodosoma granulifera, as the reference ligand. Four proteins obtained by this approach were produced for experimental evaluation. The X-ray structure of one of these proteins was determined to check for similarity of the transferred functional motif with the structure it adopts in the reference ligand. Three of these protein ligands bind the Kv1.2 channel with inhibition constants of 0.5, 1.5, and 1.6 microM. Several mutants of these designed protein ligands gave binding results consistent with the presumed binding mode. These results show that protein ligands can be designed by transferring a binding motif on a protein host selected to reproduce the functional topology of this motif, irrespective to the secondary structure supporting the functional motif, if the host protein possesses steric and electrostatic properties compatible with the binding to the target. This result opens the way to the design of protein ligands by taking advantage of the considerable structural repertoire of the Protein Data Bank.

Amino Acid Sequence↗
PubMed2004

Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogues calcipotriol and seocalcitol, receptor agonists of clinical importance. Insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modification.

The plethora of actions of 1alpha,25(OH)(2)D(3) in various systems suggested wide clinical applications of vitamin D nuclear receptor (VDR) ligands in treatments of inflammation, dermatological indication, osteoporosis, cancers, and autoimmune diseases. More than 3000 vitamin D analogues have been synthesized in order to reduce the calcemic side effects while maintaining the transactivation potency of these ligands. Here, we report the crystal structures of VDR ligand binding domain bound to two vitamin D agonists of therapeutical interest, calcipotriol and seocalcitol, which are characterized by their side chain modifications. These structures show the conservation of the VDR structure and the adaptation of the side chain anchored by hydroxyl moieties. The structure of VDR-calcipotriol helps us to understand the structural basis for for the switching of calcipotriol to a receptor antagonist by further side chain modification. The VDR-seocalcitol structure, in comparison with the structure of VDR-KH1060, a superagonist ligand closely related to seocalcitol, shows adaptation of the D ring and position of C-21 in order to adapt its more rigid side chain.

Calcitriol↗
PubMed2005

Low resolution structural study of two human HSP40 chaperones in solution. DJA1 from subfamily A and DJB4 from subfamily B have different quaternary structures.

Proteins that belong to the heat shock protein (Hsp) 40 family assist Hsp70 in many cellular functions and are important for maintaining cell viability. A knowledge of the structural and functional characteristics of the Hsp40 family is therefore essential for understanding the role of the Hsp70 chaperone system in cells. In this work, we used small angle x-ray scattering and analytical ultracentrifugation to study two representatives of human Hsp40, namely, DjA1 (Hdj2/dj2/HSDJ/Rdj1) from subfamily A and DjB4 (Hlj1/DnaJW) from subfamily B, and to determine their quaternary structure. We also constructed low resolution models for the structure of DjA1-(1-332), a C-terminal-deleted mutant of DjA1 in which dimer formation is prevented. Our results, together with the current structural information of the Hsp40 C-terminal and J-domains, were used to generate models of the internal structural organization of DjA1 and DjB4. The characteristics of these models indicated that DjA1 and DjB4 were both dimers, but with substantial differences in their quaternary structures: whereas DjA1 consisted of a compact dimer in which the N and C termini of the two monomers faced each other, DjB4 formed a dimer in which only the C termini of the two monomers were in contact. The two proteins also differed in their ability to bind unfolded luciferase. Overall, our results indicate that these representatives of subfamilies A and B of human Hsp40 have different quaternary structures and chaperone functions.

Amino Acid Sequence↗
PubMed2005

Accuracy of structure-derived properties in simple comparative models of protein structures.

The accuracy of comparative models of proteins is addressed here. A set of 12,732 single-template models of sequences of known high-resolution structures was built by an automated procedure. Accuracy of several structure-derived properties, such as surface area, residue accessibility, presence of pockets, electrostatic potential and others, was determined as a function of template:target sequence identity by comparing models with their corresponding experimental structures. As expected, the average accuracy of structure-derived properties always increases with higher template:target sequence identity, but the exact shape of this relationship can differ from one property to another. A comparison of structure-derived properties measured from NMR and X-ray structures of the same protein shows that for most properties, the NMR/X-ray difference is of the same order as the error in models based on approximately 40% template:target sequence identity. The exact sequence identity at which properties reach that accuracy varies between 25 and 50%, depending on the property being analyzed. A general characteristic of simple comparative models is that their surface has increased area as a consequence of being more rugged than that of experimental structures. This suggests that including solvent effects during model building or refinement could significantly improve the accuracy of surface properties in comparative models.

Amino Acids↗
PubMed1997

Secondary structure and shape of plasma sex steroid-binding protein--comparison with domain G of laminin results in a structural model of plasma sex steroid-binding protein.

We have analyzed the secondary structure, shape and dimensions of plasma sex steroid-binding protein (SBP) by CD, size-exclusion chromatography and electron microscopy. CD spectra show extrema at 186 nm and 216 nm characteristic for beta-sheet structures. Analysis with different algorithms indicates 15% alpha-helix, 43% beta-sheet and 10-16% beta-turn structures. An irreversible structural change is observed upon heating above 60 degrees C, which correlates with the loss of steroid-binding activity. As the SBP sequence shows similarity with domains of several multidomain proteins, including laminins, we evaluated the structure of domain G of laminin-1. The CD spectrum shows extrema at 200 nm and 216 nm. Deconvolution results in 13% alpha-helix, 32% beta-sheet and 15% beta-turn structures. Steroid-binding assays indicate that laminin and fragments thereof have no activity. Size-exclusion chromatography reveals that SBP has an extended shape and can be modeled as a cylinder with a length and diameter of 23 nm and 3 nm, respectively. This shape and the dimensions are in agreement with the appearance on electron micrographs. We propose a model for the structure of SBP in which two monomers assemble head to head with the steroid-binding site located in the center of the rod-like particle.

Amino Acid Sequence↗
PubMed2003

Quantitative structure-property relationships for predicting Henry's law constant from molecular structure.

Various models are available for the prediction of Henry's law constant (H) or the air-water partition coefficient (Kaw), its dimensionless counterpart. Incremental methods are based on structural features such as atom types, bond types, and local structural environments; other regression models employ physicochemical properties, structural descriptors such as connectivity indices, and descriptors reflecting the electronic structure. There are also methods to calculate H from the ratio of vapor pressure (p(v)) and water solubility (S(w)) that in turn can be estimated from molecular structure, and quantum chemical continuum-solvation models to predict H via the solvation-free energy (deltaG(s)). This review is confined to methods that calculate H from molecular structure without experimental information and covers more than 40 methods published in the last 26 years. For a subset of eight incremental methods and four continuum-solvation models, a comparative analysis of their prediction performance is made using a test set of 700 compounds that includes a significant number of more complex and drug-like chemical structures. The results reveal substantial differences in the application range as well as in the prediction capability, a general decrease in prediction performance with decreasing H, and surprisingly large individual prediction errors, which are particularly striking for some quantum chemical schemes. The overall best-performing method appears to be the bond contribution method as implemented in the HENRYWIN software package, yielding a predictive squared correlation coefficient (q2) of 0.87 and a standard error of 1.03 log units for the test set.

Chemical Phenomena↗
PubMed2003

Structural, chemical topological, electrotopological and electronic structure hypotheses.

The first important hypothesis in the prediction of properties of synthesized molecules is the structural hypothesis. In the study of drug-receptor interactions, the case where the three-dimensional structure of the receptor is known allows the application of molecular simulation and energy calculations to estimate the binding affinity for a proposed series of compounds. The chemical topological hypothesis permits the description of molecular structures without using concepts such as force or energy. These notions would not be as dominant as supposed since they should be able to be deduced from topology. Although topological descriptors are able to describe specific physicochemical properties, there is no any mechanistic interpretation for topological descriptors, but they can be considered as essential magnitudes, able to describe molecular structure as an alternative and independent approach, since they allow an algebraic description of the structure itself. The hypothesis is based on the ability of topological descriptors for the prediction of virtually any kind of structure-related macroscopic property, and to design directly new compounds showing predetermined properties. It is possible to design new lead drugs ignoring the explicit mechanism of action, but only by topological similarity with other active compounds. This is a basic difference with all the conventional methods based on the use of physical variables, which need to know the specific drug-receptor interaction. The electrotopological hypothesis may be considered a synthesis of both previous hypotheses. Finally, the electronic structure hypothesis is formulated.

Electrons↗
PubMed1996

Analysis of topological and nontopological structural similarities in the PDB: new examples with old structures.

We have developed a new method and program, SARF2, for fast comparison of protein structures, which can detect topological as well as nontopological similarities. The method searches for large ensembles of secondary structure elements, which are mutually compatible in two proteins. These ensembles consist of small fragments of C alpha-trace, similarly arranged in three-dimensional space in two proteins, but not necessarily equally-ordered along the polypeptide chains. The program SARF2 is available for everyone through the World-Wide Web (WWW). We have performed an exhaustive pairwise comparison of all the entries from a recent issue of the Protein Data Bank (PDB) and report here on the results of an automated hierarchical cluster analysis. In addition, we report on several new cases of significant structural resemblance between proteins. To this end, a new definition of the significance of structural similarity is introduced, which effectively distinguishes the biologically meaningful equivalences from those occurring by chance. Analyzing the distribution of sequence similarity in significant structural matches, we show that sequence similarity as low as 20% in structurally-prealigned proteins can be a strong indication for the biological relevance of structural similarity.

Algorithms↗
PubMed2000

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant.

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.

Animals↗
PubMed2005

Helix packing motif common to the crystal structures of two undecapeptides containing dehydrophenylalanine residues: implications for the de novo design of helical bundle super secondary structural modules.

De novo designed peptide based super secondary structures are expected to provide scaffolds for the incorporation of functional sites as in proteins. Self-association of peptide helices of similar screw sense, mediated by weak interactions, has been probed by the crystal structure determination of two closely related peptides: Ac-Gly1-Ala2-Delta Phe3-Leu4-Val5-DeltaPhe6-Leu7-Val8-DeltaPhe9-Ala10-Gly11-NH2 (I) and Ac-Gly1-Ala2-DeltaPhe3-Leu4-Ala5-DeltaPhe6-Leu7-Ala8-DeltaPhe9-Ala10-Gly11-NH2 (II). The crystal structures determined to atomic resolution and refined to R factors 8.12 and 4.01%, respectively, reveal right-handed 3(10)-helical conformations for both peptides. CD has also revealed the preferential formation of right-handed 3(10)-helical conformations for both molecules. Our aim was to critically analyze the packing of the helices in the solid state with a view to elicit clues for the design of super secondary structural motifs such as two, three, and four helical bundles based on helix-helix interactions. An important finding is that a packing motif could be identified common to both the structures, in which a given peptide helix is surrounded by six other helices reminiscent of transmembrane seven helical bundles. The outer helices are oriented either parallel or antiparallel to the central helix. The helices interact laterally through a combination of N--H...O, C--H...O, and C--H...pi hydrogen bonds. Layers of interacting leucine residues are seen in both peptide crystal structures. The packing of the peptide helices in the solid state appears to provide valuable leads for the design of super secondary structural modules such as two, three, or four helix bundles by connecting adjacent antiparallel helices through suitable linkers such as tetraglycine segments.

Amino Acid Motifs↗
PubMed2004

Raman spectroscopic characterization of drying-induced structural changes in a therapeutic antibody: correlating structural changes with long-term stability.

We characterized the secondary structure of a therapeutic recombinant humanized monoclonal antibody (rhuMAb), formulated with different concentrations of sucrose, trehalose, and histidine and in solution, lyophilized, and spray-dried states. Quantitative secondary structure estimates were obtained using amide I band Raman spectroscopy and a previously developed spectral deconvolution procedure. On lyophilization or spray drying in the absence of sugar, the antibody underwent significant structural perturbation. The beta-sheet content decreased with corresponding gain in the turn and unordered content. With increasing amount of sucrose or trehalose, the extent of structural perturbation decreased. Eventually, at sugar-to-protein molar ratios of > or =360, almost complete structural preservation was observed. Histidine also protected the antibody against lyophilization-induced structural changes. The extent of structural perturbation immediately after lyophilization or spray drying exhibited good correlation with the rate of aggregation for the antibody during long-term storage under accelerated conditions. The results demonstrate that amide I band Raman spectroscopy could be a quick and reliable way to screen excipients and their concentrations during lyophilized or spray dried formulation development.

Antibodies, Monoclonal↗
PubMed1993

Modeling the structure of Pyrococcus furiosus rubredoxin by homology to other X-ray structures.

The three-dimensional structure of rubredoxin from the hyperthermophilic archaebacterium, Pyrococcus furiosus, has been modeled from the X-ray crystal structures of three homologous proteins from Clostridium pasteurianum, Desulfovibrio gigas, and Desulfovibrio vulgaris. All three homology models are similar. When comparing the positions of all heavy atoms and essential hydrogen atoms to the recently solved crystal structure (Day, M. W., et al., 1992, Protein Sci. 1, 1494-1507) of the same protein, the homology model differ from the X-ray structure by 2.09 A root mean square (RMS). The X-ray and the zinc-substituted NMR structures (Blake, P. R., et al., 1992b, Protein Sci. 1, 1508-1521) show a similar level of difference (2.05 A RMS). On average, the homology models are closer to the X-ray structure than to the NMR structures (2.09 vs. 2.42 A RMS).

Amino Acid Sequence↗