Correlation of secondary structure with biological activity for a leader peptide: circular dichroism-derived structure and in vitro biological activities of preproparathyroid hormone peptide and its analogs.
Leader or signal sequences are specialized domains within precursor proteins which serve an essential role in interacting with the cellular secretory apparatus to enable intracellular transport and secretion of proteins. Despite many differences in primary amino acid sequences, signal domains interact with a common set of intracellular components, presumably because the signal sequences share an overall conformational similarity. In a few instances, mutant signal peptides from prokaryotes have been studied and their structures correlated with function (export) in vivo. A series of analogs of the precursor-specific region of preproparathyroid hormone have been prepared which contain substitutions of either proline or a charged amino acid within the hydrophobic core. These synthetic "mutants" have previously been evaluated in several in vitro assays to determine their functionality with regard to protein secretion and suitability as substrates for signal peptidase. The secondary structural content of each peptide, as well as the native sequence and sulfur-free analog, was determined in aqueous and nonaqueous conditions by circular dichroism (CD) as a function of time. The structures obtained were correlated with in vitro bioactivities. Unlike the findings or previous CD studies, all the peptides examined here had low to undetectable alpha-helical content in both aqueous and nonaqueous buffers. The unsubstituted and sulfur-free analogs had high (80-85%) beta-structure in aqueous conditions which was reduced to approximately 30% in nonaqueous solvent. The proline- and charged-substituted peptides contained about half the beta-structure content (35-55%) in aqueous buffer; in nonaqueous solvent their structure was similar to the unsubstituted peptides. The structure-activity correlates found were as follows: a high degree of structure (aqueous conditions) correlated with interaction with signal recognition particle and substrate suitability for signal peptidase; a low degree of structure (nonaqueous environment) correlated with activity in the translocation assay.
Caulfield
M P
MP
Department of Biological Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Park
K
K
Rosenblatt
M
M
Fasman
G D
GD
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism; D066298:In Vitro Techniques; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D008565:Membrane Proteins; D010281:Parathyroid Hormone; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011506:Proteins; C019056:preproparathormone; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase