The two-domain NK1 fragment of plasminogen: folding, ligand binding, and thermal stability profile.
The two-domain fragment N+K1 (rNK1) [Glu(1)-Glu(163)] of human plasminogen was expressed in E. coli as a hexahistidine-tagged fusion protein and chromatographically purified. The (1)H NMR spectrum supports proper folding of the K1 component within the refolded rNK1 construct (rNK1/K1). The functional properties of rNK1/K1 were investigated via intrinsic fluorescence titration with kringle-specific omega-aminocarboxylic acid ligands. The affinities closely match those previously measured for the isolated K1, which indicates that the N-domain does not significantly affect the interaction of ligands with the lysine binding site of K1. Far-UV CD spectra recorded for the N-domain suggest conformational plasticity and flexibility for the module. Two classes of spectra, referred to as types A and B, were identified with the type A spectrum reflecting a higher secondary structure content than that estimated for the type B spectrum. Subtracting the CD spectrum of rK1 from that of rNK1 yields a spectrum (Delta) which reflects the conformation of the N-domain within the rNK1 construct (rNK1/N). Delta resembles the type A spectrum, suggesting that rNK1/N adopts a relatively more ordered conformation, stabilized by the adjacent rNK1/K1 domain. In contrast, thermal unfolding curves determined via CD indicate that the rNK1/N slightly lowers the melting temperature (T(m)) of rNK1/K1. Independence of the two domains within rNK1 was tested by monitoring the thermal unfolding of rNK1/K1 when in the presence of the kringle-specific ligand AMCHA, which left the rNK1/N T(m) essentially unaffected, while increasing that of the rNK1/K1 by approximately 10 degrees C.
Douglas
Justin T
JT
Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
von Haller
Priska D
PD
Gehrmann
Marion
M
Llinás
Miguel
M
Schaller
Johann
J
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018076:DNA, Complementary; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature
D018076:DNA, Complementary; D008024:Ligands; D010446:Peptide Fragments; D010958:Plasminogen