Structural significance of sequence variability in antibody complementarity-determining regions.
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Amino Acid Sequence↗
Biomedical subjects
C Abergel
Publications and source records attributed to C Abergel.
Crystallographic studies and primary structure of the antitumor monoclonal CC49 Fab'.
The Fab' of CC49, a murine monoclonal antibody directed against the human tumor-associated antigen TAG-72 has been crystallized. The crystals are monoclinic, space group P(2)1 with cell parameters a = 115.6 A, b = 116.4 A, and c = 70.3 A; beta = 97.8 degrees. The size of the unit cell is compatible with four Fab' molecules in the asymmetric unit. The Fab molecules are related by two approximately perpendicular pseudo-2-fold axes. One pseudo-2-fold axis is parallel to the crystallographic 2-fold axis and was found by inspection of the Harker section of the native Patterson map; the other was found by a self rotation function. The primary structures of the variable regions of the CC49 antibody light and heavy chains have been determined and are compared with those of the related antitumor antibody B72.3.
Amino Acid Sequence↗
Isoform purification of gastric lipases. Towards crystallization.
Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.
Animals↗
Systematic use of the incomplete factorial approach in the design of protein crystallization experiments.
We have used the Incomplete Factorial Approach (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) in conjunction with the program Cristal (Roussel, A., Serre, L., Frey M., and Fontecilla-Camps, J. (1990) J. Crystal Growth 106, 405-409) to crystallize six different proteins. We were able to obtain crystals and to identify the critical factors for crystallization for each of these six proteins. In some of the cases, we succeeded on the first try while using only minute amounts of protein. This study proves that the Incomplete Factorial Approach is a powerful tool in identifying the factors that need to be varied to achieve crystallization. Single crystals of adequate size were obtained for all the proteins reported here, although some did not diffract well enough to be studied by x-ray diffraction methods; the asymmetric units of these latter crystals contain a large metric units of these latter crystals contain a large number of molecules, which is most likely due to the presence of significant amounts of carbohydrate in the proteins.
Animals↗
Crystallization and preliminary X-ray diffraction data of the Fab fragment of a monoclonal antibody against apamin, a bee venom neurotoxin.
Fab fragments of anti-apamin monoclonal antibodies have been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group P21 with cell dimensions a = 99.0 +/- 0.3 A, b = 137.1 +/- 0.4 A, c = 76.0 +/- 0.2 A and beta = 92.9 +/- 0.9 degrees. They most likely contain four molecules in the asymmetric unit (Vm = 2.39 A3/Da). The possibility of the existence of non-crystallographic symmetry is discussed.
Antibodies, Monoclonal↗
A strong propensity toward loop formation characterizes the expressed reading frames of the D segments at the Ig H and T cell receptor loci.
A compilation of murine and human Ig H and TcR beta D segment sequences was used to estimate the relative usage of the various reading frames and to look for associated sequence patterns. We confirm a strong bias in the expression of the Ig H D segments, with more than 90% (murine) and 85% (human) expressed peptides resulting from a preferred reading frame. Remarkably, 86% (mouse) and 90% (human) of those peptides contain at least one glycine residue. All but one of the atypical preferred D peptides contain serine or proline residues and are found in the immediate vicinity of glycine residues provided by specific JH segments. The presence of tyrosine residues is also a characteristic feature of expressed reading frames in both mouse (75%) and human (90%). These results suggest that the constraints of forming a flexible loop within the third complementarity-determining region, is a factor in the preference for a particular reading frame in Ig H D. For the TcR beta D segments, glycine is specified in most reading frames, and no significant preference is observed.
Amino Acid Sequence↗
Crystallization and preliminary X-ray study of a recombinant cutinase from Fusarium solani pisi.
Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized at 20 degrees C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol at 15 to 20%. They are monoclinic, with space group P2(1) and cell dimensions a = 35.1 A, b = 67.4 A, c = 37.05 A and beta = 94.0 degrees; they diffract beyond 1.5 A resolution. The asymmetric unit contains one molecule of 22,000 Da (Vm = 1.98 A3/Da; 38% water).
Carboxylic Ester Hydrolases↗
Crystallization and preliminary X-ray study of AaH IT2, and insect-specific toxin from the scorpion Androctonus australis Hector.
An insect-specific toxin from the venom of the scorpion Androctonus australis Hector has been crystallized. The crystals are orthorhombic, space groups P2(1)2(1)2(1), with cell dimensions a = 66.4 A, b = 52.5 A and c = 36.1 A. Calculations based on the unit cell volume and toxin molecular mass suggest that there are two molecules in the asymmetric unit.
Animals↗
X-ray crystal structure determination and refinement at 1.9 A resolution of isolectin I from the seeds of Lathyrus ochrus.
Orthorhombic crystals of isolectin I (LOLI) from the seeds of Lathyrus ochrus were first obtained during the STS 29 space shuttle mission. Subsequently, isostructural crystals were also obtained in the laboratory. They belong to the space group P2(1)2(1)2, with cell dimensions a = 135.84 A, b = 63.12 A and c = 54.54 A with one functional entity, a dimer, in the asymmetric unit (Vm = 2.2 A3/Da). The three-dimensional structure of LOLI, which was solved by the molecular replacement method using a 3 A resolution model of pea lectin, has subsequently been refined by using crystallographic data between 8.0 A and 1.9 A resolution, coupled to molecular dynamics and energy minimization techniques. The conventional R-factor is 0.185 for Fo greater than 1 sigma(Fo). The final model includes 220 well-defined water molecules and has root-mean-square deviations from ideal bond distances and angles of 0.004 A and 3 degrees, respectively. Only slight conformation differences have been found between the two alpha beta monomers. The molecular structure of LOLI, the first to be determined from the genus Lathyrus, is very similar to those of concanavalin A, pea lectin and favin. Differences are confined to the loop regions and beta-chain termini. Comparison of equivalent C alpha atom positions between our final model and the pea lectin structure shows slight differences in the association of the two monomers, which are probably due to the different environments in the crystals. The root-mean-square deviation between C alpha atoms of LOLI and pea lectin is 0.40 A. The metal binding sites are very similar in pea lectin, concanavalin A and LOLI. The sugar-binding sites of LOLI are occupied by four well-ordered water molecules each. The cleavage site for one of the monomers is specially well defined in the final electron density map: the amino group of Glul (alpha) seems to form a salt bridge with the carboxylate group of the terminal Asn181 (beta). A detailed analysis of the difference in crystal packing contacts between pea lectin and LOLI shows that, as might be expected, several of the intermolecular interactions are mediated by residues that correspond to substitutions in the LOLI amino acid sequence.
Amino Acid Sequence↗
Conformational analysis of T immunogenic peptides by circular dichroism spectroscopy.
The structure of two T-immunogenic peptides, one from the gag p24 protein of the human immunodeficiency virus, the other from the 11.1 gene product of Plasmodium falciparum, was studied by circular dichroism spectroscopy in various pH and solvent conditions. Although both sequences are predicted to adopt an alpha-helical conformation and one of them is a repeat of a perfect alpha-amphipathic sequence pattern, these two peptides exhibit a strong propensity to adopt an extended, turn or aperiodical conformation in solution.
Circular Dichroism↗