Conformational stability studies of the pleckstrin DEP domain: definition of the domain boundaries.
Pleckstrin is the major substrate of protein kinase C in platelets. It contains at its N- and C-termini two pleckstrin homology (PH) domains which have been proposed to mediate protein-protein and protein-lipid interactions. A new module, called DEP, has recently been identified by sequence analysis in the central region of pleckstrin. In order to study this module, several recombinant polypeptides corresponding to the DEP module and N- and C-termini extended forms have been expressed. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques, the domain boundaries have been determined that yield a soluble and folded pleckstrin DEP domain. This comprises 93 amino acids with an alpha/beta fold in agreement with secondary structure predictions. Stability studies indicate that the regions surrounding the DEP domain do not contribute to its stability suggesting that the phosphorylation sites at S113, T114 and S117 are in an unstructured region. Identification of the regions of pleckstrin that are folded shall facilitate determination of its structure and function.
Kharrat
A
A
EMBL, Meyerhofstr. 1, D-69012 Heidelberg, Germany.
Millevoi
S
S
Baraldi
E
E
Ponting
C P
CP
Bork
P
P
Pastore
A
A
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010750:Phosphoproteins; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D001798:Blood Proteins; D010455:Peptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; C039077:platelet protein P47