Biomedical subjects
Publications and source records attributed to E A Padlan.
Immunoglobulin sequences were compared by using a technique that takes into account the dissimilarity in physicochemical properties of amino acids. Exterior residues showed greater structural variability than interior residues. High structural variability was found at positions known from crystallographic studies to be involved in hapten binding.
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Crystals of solid phase-derived semisynthetic ribonuclease-S' were prepared and compared with those for native ribonuclease-S' and -S. The semisynthetic species used was the noncovalent complex of synthetic fragment-(1-20), corresponding to residues 1 through 20 of bovine pancreatic ribonuclease-A (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22), and native ribonuclease-S-(21-124); the fragment containing residues 21 through 124 of ribonuclease-A. This semisynthetic complex was completely active enzymatically, was homogeneous as judged by polyacrylamide gel electrophoresis, and had no greater than trace amounts of excess ribonuclease-s(21-124) as judged by affinity chromatography. Crystallization of both semisynthetic and native ribonuclease-s' at pH 5.3 resulted in well-formed crystallseater than trace amounts of excess ribonuclease-S-T21-124) as judged by affinity chromatography. Crystallization of both semisynthetic and native ribonuclease-S' at pH 5.3 resulted in well-formed crystals with the symmetry of space group P3121 and unit cell dimensions a=b=44.82, c=97.3 A. This crystal form corresponds to the Y form of native ribonuclease-S previously reported [Wyckoff et al. (1967) J. Biol. Chem. 242, 3749-3753]. X-ray diffraction patterns of the crystals were indistinguishable, indicative of the structural identity of semisynthetic and native ribonuclease-S'.
The crystal structures of acid metmyoglobin and deoxy cobalt(II)mesoporphyrin IX myoglobin were compared by a difference Fourier analysis at 2.5 A resolution. No large differences in protein conformation were observed. The greatest density of structural differences was found in the heme region. There was a loss of the histidine-bound sulfate ion and of the metal-bound water molecule, as well as a shift in the position of the prosthetic group with associated changes in the adjacent globin. The structural changes resulting from the substitution of ethyl for the vinyl side chains of the porphyrin were clearly observed. There was also a suggestion of a conformational change of the porphyrin ring. It was not clear whether there was any change of the metal position relative to the porphyrin plane or proximal histidine.
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A comparison of various variable immunoglobulin domains demonstrates that structural variability parallels the observed frequency of amino-acid substitutions. The similarity of the structural frameworks is in good agreement with the evolutionary relationships of the chains as derived from sequence analysis.
A comparison of five constant region sequences of human and mouse k and lambda immunoglobulin chains has been undertaken in order to reveal sequence homologies and evolutionary relationships. Simultaneously, a comparison with the three-dimensional structure of one mouse k-chain (McPC 603) has suggested structural reasons why many of the residues are invariant or conserved along k versus lambda lines. There are a number of residues that have remained invariant despite exposed positions for reasons that do not apppear to be connected with the folding of the CL domain.
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The structure of the Fab of McPC 603, a mouse myeloma protein with phosphorylcholine binding activity, has been determined to 3.1-A resoltuion. The four domains are found to be structurally similar with a well-defined double-layer structure. A large cavity exists at one end of the fragment, the walls of which are formed exclusively of hypervariable residues. Phosphorylcholine binds in this cavity and forms specific interactions with several well-defined amino-acid side chains of the protein. The hapten is bound asymmetrically and interacts more with the heavy chain than with the light chain.
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Fab-fragments of several phosphorylcholine-binding mouse-myeloma proteins have been prepared by pepsin digestion; two of these, MOPC 167 and McPC 603, gave large crystals from ammonium sulfate solutions. The Fab-fragment from MOPC 167 crystallizes in a hexagonal space group, but does not diffract to a resolution greater than about 8 A. In contrast, McPC 603 crystals (space group P6(3)) diffract to about 2.7 A. An isotopic double-labeling technique was developed that demonstrated that the 603 crystals bind 1 mol of hapten per mol of Fab-fragment, but with a binding constant significantly lower than that observed in solution. The findings indicate that a three-dimensional model of this homogeneous antigen-binding immunoglobulin can be constructed. Accordingly, a search for heavy-atom derivatives and determination of the primary structure are in progress.
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It is proposed to reduce the immunogenicity of allogeneic antibody variable domains, while preserving ligand-binding properties, by reducing their antigenicity through replacement of the exposed residues in the framework regions which differ from those usually found in host antibodies. The results of a comparison of representative murine antibody sequences with those of human origin suggest that the number of residues that need to be replaced to "humanize" those antibodies could be small.