CONFORMATIONAL STUDIES ON ANALOGS OF RECOMBINANT PARATHYROID-HORMONE AND THEIR INTERACTIONS WITH PHOSPHOLIPIDS

Through the use of oligonucleotide-directed mutagenesis we have genera ted variants of a recombinant human parathyroid (PTH) hormone-(1-34)-h omoserine (RPTH) in which a positively charged residue (Arg or Lys), a negatively charged residue (Glu), or a neutral residue (Gly) has been substituted at every position throughout the peptide. These 106 PTH a nalogs have been tested for their ability to stimulate cAMP production in the rat osteosarcoma cell line, UMR106. Analysis of these peptides led to the construction of several analogs containing multiple substi tutions at sites of potential structural importance. Several of these analogs were shown to have 3-5-fold enhanced activity and receptor aff inity. Circular dichroism (CD) and lipid binding studies were then per formed on these analogs. Circular dichroism demonstrates enhanced heli cal content in the presence of lipid vesicles, particularly anionic li pid. The [Arg(15,19,22),Lys(29)]RPTH (+6RPTH) analog requires higher c oncentrations of trifluoroethanol to attain enhanced helicity. The int rinsic tryptophan fluorescence of the peptides are blue shifted more i n the presence of the anionic lipid dimyristoyl phosphatidylglycerol ( DMPG) than with the zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), Effects of the peptides on the phase transition behavior of D MPC shows that +6RPTH has less effect on the lipid than does RPTH. Thi s difference in lipid interaction is also exhibited with isothermal ti tration calorimetry, in which RPTH reacts exothermally with DMPG, whil e +6RPTH shows little or no heat change, The pH dependence of binding of the hydrophobic probe 1,1'-bis(4-anilino)naphthalene-5,5'-trisulfon ic acid, also shows a difference in exposure of hydrophobic sites betw een RPTH and +6RPTH. The +6RPTH has about a 5-fold greater affinity fo r receptor binding, We suggest that this enhanced activity is a conseq uence of the altered lipid interaction of +6RPTH, combined with increa sed conformational flexibility, particularly in the carboxyl-terminal region of the molecule.