Human monoclonal cryoimmunoglobulins. I. Molecular properties of IgG3 kappa (Jir protein) and the cryo-coprecipitability of its molecular fragments by papain

The temperature-dependent precipitability of a monoclonal IgG3 kappa cryoimmunoglobulin (Jir) without known antibody activity is shown to be affected by various physico-chemical factors, such as protein concentration, pH value and NaCl concentration. The molecular properties characterizing this protein (carbohydrate and amino acid compositions, peptide constitutions and susceptibility to enzymatic proteolysis) are described. The cryoprecipitability of the protein was completely lost upon papain hydrolysis, and none of the isolated fragments, Fab-Fc, Fc, and Fab, showed any precipitating activity. In the cryo-coprecipitation assay using the 125I-labeled fragments, it was demonstrated that the association activity with intact Jir protein was still retained on the Fab-Fc and Fc fragments, but not on the Fab fragment. The evidence suggests that a specific interaction may be involved in the primary intermolecular association required to form the cryoprecipitate at temperatures below the critical point, and that one of the pairing sites resides on the Fc portion of the protein molecule.     

Hydration of compact and extended forms of the pFh fragments of myeloma IgG3

It has been shown by scanning microcalorimetry and densitometry that the partial specific heat and the partial specific volume of pFh fragments of two myeloma IgG3 increase during breakdown of the tertiary structure, while the secondary structure—left-handed poly-L-proline II double helix—does not change. This effect may be explained by a high degree of hydration, which increases upon globule decompactization because of enhanced solvent accessibility of the peptide groups of the helix.     

Short fragments of a protein globule with predominant conformation

It has been found that 1500 tetrapeptides out of 160000 possible combinations occurring in proteins exhibit preference for particular conformational states. Most conformationally stable tetrapeptides obtained in the analysis of a sampling containing 706 proteins are in the alpha-helical form. The features of the amino acid composition of conformationally stable oligopeptides have been studied.     

Characterization of two species of Fc fragments obtained from rat IgG2a by prolonged papain digestion.

Prolonged papain digestion of rat IgG2a produced two molecular species of Fc fragments, termed Fc(I) and Fc(II). Studies by gel filtration chromatography and polyacrylamide gel electrophoresis in SDS/urea indicated that the two subunit polypeptide chains in each Fc preparation were associated by non-covalent bonds only. By analytical ultracentrifugation Fc(I) was found to have a m.w. of 47,100 and a sedimentation coefficient of 4.08S. Fc(II) had a m.w. of 39,800 and a sedimentation coefficient of 3.83S. The m.w. for the subunit chains of Fc(I) and Fc(II) were 25,300 and 20,300, respectively, as determined by analytical ultracentrifugation under dissociating conditions. Calculation of the frictional coefficient ratios indicated that both Fc fragments possessed compact globular structures. The difference in size between these two Fc fragments probably was due to a loss of some carboxy-terminal residues in Fc(II). Both Fc fragments possessed nearly identical amino-terminal amino acid sequences. Papain cleavage occurred primarily between residues 233/234 and 234/235. The carbohydrate compositions of the two species of Fc fragments were similar. It was concluded that under acid and reducing conditions papain cleavage of rat IgG2a occurred to the carboxy-terminal side of the hinge region. Prolonged papain digestion led to secondary attack in the carboxy-terminal end of the CGAMMA3 domain at an unidentified site, or sites, producing a stable second species of Fc fragments.     

Ponticulin is an atypical membrane protein

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha- helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).     

The interaction of ribosomal protein L16 and its fragments with tRNA

Two large proteolytic fragments of Escherichia coli 50 S ribosomal subunit protein L16 were generated by limited hydrolysis with chymotrypsin (missing 9 N-terminal amino acids) and trypsin (missing 16 N-terminal amino acids). It was found that while intact L16 and its chymotryptic fragment both interact with tRNA (Kd = 5.4 x 10(-7) M), the tryptic fragment does not. These results are interpreted in terms of possible significance of the residues 10-16 in the peptidyl transferase activity.     

The prediction of transmembrane protein sequences and their conformation: an evaluation

The prediction of transmembrane protein sequences can be performed with reasonable accuracy. However, the prediction of their conformation leaves room for significant improvement.     

Folding of protein fragments: conformational and biological studies on thioredoxin and its fragments

Thioredoxin can be cleaved enzymatically into the two fragments (1–73) and (74–108) and chemically into two different ones (1–37) and (38–108). In this paper, the conformational properties of the short fragment (1–37) are reported and compared with those of the larger fragment (1–73). Using mainly circular dichroism (CD), it is shown that the (1–37) fragment, which contains the active disulfide unit center, is present as an unordered structure in the neutral pH range, but assumes a rigid folding at pH values below 6. The form of the CD spectrum is very similar to that of the complete native protein, and to that of the folded (1–73) fragment. The possible mechanisms for refolding of the short fragment are discussed.     

Mussel glue protein has an open conformation

Both native glue protein from marine mussels and a synthetic nonhydroxylated analog were analyzed by far-uv CD under a variety of conditions. Analysis of the CD spectra using various models strongly suggest a primarily random coil structure for both forms of the protein, a fact also supported by the absence of spectral change for the glue protein upon dilution into 6 M guanidine hydrochloride. The nonhydroxylated analog, which consists of 20 repeats of the peptide sequence Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys, was further characterized by enzyme modification using mushroom tyrosinase. Enzymatic hydroxylation of tyrosines was found to be best fit by a model containing two rate constants, 5.6 (+/- 0.6) X 10(-3) and 7.2 (+/- 0.3) X 10(-2) min-1. At equilibrium, HPLC analysis of digests showed nearly 100% conversion of Tyr-9 and only 15 to 35% conversion of Tyr-5. The Chou and Fasman rules for predicting structure were applied to the repeat sequence listed above. The rules predict the absence of alpha helix and beta pleated sheets in the structure of this peptide. On the other hand, beta turns are predicted to be present with Tyr-5 being in the region of highest probability. These data suggest that the protein in solution has only a small amount of secondary structure.     

The conformation properties and conformation stability of streptokinase and its polymer derivative

The properties and conformational stability of the proteinaceous activator of fibrinolysis--native streptokinase--and its derivative obtained by modification with a linear hydrophilic copolymer based on N-vinylpyrrolidone, were studied by the circular dichroism method. It was shown that polymeric modification of streptokinase had no effect on the secondary structure, while the conformational stability of the modified protein to urea was higher than that of the native one. Studies on thermal stability of both native and modified forms of streptokinase showed that the inactivation rate was lower in the modified form as compared to the native one.