Peptide antisera as sequence-specific probes of protein conformational transitions: calmodulin exhibits calcium-dependent changes in antigenicity.
Local changes in conformation between the calcium-saturated and calcium-free forms of calmodulin were monitored using antisera to four peptides corresponding to three helical regions of the calcium-saturated protein. The N-terminal helix was monitored using antiserum to residues 9-19, calmodulin-(9-19); the C-terminal helix using antiserum to residues 141-148, calmodulin-(141-148); and the long central helix with antisera to residues 68-79 and 80-92, calmodulin-(68-79) and -(80-92). Crossreactivities of peptide antisera with calmodulin (either in the presence or absence of calcium) were determined using solution-phase and solid-phase immunoassays. When examined by the fluid-phase assay, all four peptides elicited antibody that precipitated radiolabeled apocalmodulin but not the calcium-saturated form of the protein. Similarly, when calmodulin was immobilized on a solid-support, only the calcium-free form readily bound the antibodies to calmodulin-(80-92) and -(141-148). In addition, the crossreactivity of antiserum to calmodulin-(68-79) with calcium-saturated calmodulin in solid phase was reduced by approximately equal to 40% relative to reactivity with apocalmodulin. According to the x-ray crystal structure of Ca2+-saturated calmodulin and the antigenic reactivity of calmodulin for the peptide antisera in the absence of calcium, the regions of the protein monitored by these antisera are exposed to the surface in both conformational states and probably accessible to specific antibodies. The apparent preference of peptide antibodies for one conformation of the molecule suggests that changes in the conformation of calmodulin occur in cognate sequences that are transformed by calcium from antigenic, flexible structures to less antigenic, relatively helical structures. Peptide antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes providing the cognate sequence is sterically accessible to antibody in both states but antigenic in only one.
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000276:immunology* / Q000378:metabolism; D002417:Cattle; D011232:Chemical Precipitation; D004533:Egtazic Acid; D000939:Epitopes; D009842:Oligopeptides / Q000276:immunology; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D002147:Calmodulin; D000939:Epitopes; D009842:Oligopeptides; D004533:Egtazic Acid; D002118:Calcium