PubMed · 4680722
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M C Tobin. 1972. Raman spectroscopy.. https://doi.org/10.1016/s0076-6879(72)26025-6
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The phosphoprotein (P) of vesicular stomatitis virus was previously shown to assemble into a homomultimer upon phosphorylation by casein kinase II. It thus acquired transcriptional activity, including the ability to bind to the other two transcriptional components, the polymerase L and the N-RNA template. This multimer has now been found to be a trimer using a His-tag dilution method. Trimer stability was assessed using a variation of this method, by measuring the rate of exchange of monomers between preformed tagged and untagged trimers at different values of pH and ionic strength. Exchange rates increased with increasing ionic strength and were similar at pH 6, 8, and 10, but the trimer was completely dissociated at pH 4. This suggests that the trimer is stabilized by electrostatic interactions, probably involving carboxylate and guanidino groups. Addition of viral L protein stabilized the P trimers, completely preventing subunit exchange under transcription conditions. The association constants (Kass) for trimerization of partially active D and A substitution mutants were also determined by His-tag dilution and found to correlate well with transcriptional activity, further confirming that the active species is the trimer. Circular dichroism spectra were identical for phosphorylated and unphosphorylated wild-type P protein and for D and A mutants known to be predominantly trimeric and monomeric, respectively.
Di-leucine motifs have been implicated in the internalization or degradation of many membrane proteins. The epidermal growth factor receptor (EGFR) contains two di-leucine residues at 658 (TLRRLLQER) and 679 (NQALLRIL). To determine the role of these di-leucine motifs in regulating EGF receptor expression, activity, or ligand-induced degradation, the di-leucine residues at positions 658 or 679 were mutated to di-alanine residues, and the mutant receptors were stably expressed in CHO cells. The results indicate that mutation of either di-leucine motif generates and promotes cell surface expression of carboxy-truncated EGF receptors (M(r) 120, 140 kDa) that do not undergo EGF-induced autophosphorylation or degradation. In contrast, full-length EGF receptors (170 kDa) containing di-alanine substitutions resemble wild type receptors in that they respond to EGF by autophosphorylation, their tyrosine kinase activity is inhibited by protein kinase C, and they are degraded. The level of autophosphorylation of the 170 kDa mutant receptors and EGF-induced tyrosine phosphorylation of other cellular proteins is lower than that of the wild type receptor, consistent with formation of kinase-inactive heterodimers between the truncated and full-length mutant receptors. These results demonstrate that removal of either of the di-leucines leads to generation of inactivating carboxy-truncated receptors, suggesting that the two di-leucine motifs within the juxtamembrane region of the EGFR are important for ensuring normal receptor expression.
We determined the extent to which additional binding energy could be achieved by diversifying the complementarity determining regions (CDRs) located in the center of the antibody combining site of C6.5, a human single-chain Fv (scFv) isolated from a non-immune phage library which binds the tumor antigen c-erbB-2. CDR3 of the light (V(L)) and heavy (V(H)) chain variable region of C6.5 were sequentially mutated, the mutant scFv displayed on phage, and higher affinity mutants selected on antigen. Mutation of V(L) CDR3 yielded a scFv (C6ML3-9) with a 16-fold lower Kd (1.0 x 10(-9) M) than C6.5. Due to its length of 20 amino acids, four V(H) CDR3 libraries of C6ML3-9 were constructed. The greatest increase in affinity from a single library was ninefold (Kd = 1.1 x 10(-10) M). Combination of mutations isolated from separate V(H) CDR3 libraries yielded additional ninefold decreases in Kd, resulting in a scFv with a 1230-fold increase in affinity from wild-type C6.5 (Kd = 1.3 x 10(-11) M). The increase in affinity, and its absolute value, are comparable to the largest values observed for antibody affinity maturation in vivo or in vitro and indicate that mutation of V(L) and V(H) CDR3 may be a particularly efficient means to increase antibody affinity. This result, combined with the location of amino acid conservation and substitution, suggests an overall strategy for in vitro antibody affinity maturation. In addition, the affinities and binding kinetics of the single-chain Fv provide reagents with potential tumor targeting abilities not previously available.