Proton Overhauser experiments on kringle 4 from human plasminogen. Implications for the structure of the kringles' hydrophobic core

Abstract

1H-NMR Overhauser experiments at 300 and 600 MHz have been implemented on the isolated kringle 4 fragment of human plasminogen. This study shows that Leu 46 and Leu 77 CH 3 δ,δ′ groups, as well as two threonine CH 3 γ and a methionine S-CH ϵ (probably Met 48 groups, are in efficient dipolar contact with histidine and aromatic side-chains. In particular, the experiments reveal that of the two Leu 46 CH 3 δ,δ′ groups, one is in efficient contact with tryptophan (Trp 25 and Trp 62 indole rings while the other interacts with a tyrosine (probably Tyr 41) phenol. Leu 46 appears also to be close to an Ala CH 3 β group. Such a hydrophobic cluster appears to be contiguous to Trp 72, hence to Arg 71, residues that are through to be part of the lysine-binding site. Acid-base titration experiments show that the buried methionine S-CH 3 ϵ group senses a neighboring ionizable group pK ∗ 1 = 3.76 , suggesting presence of a carboxyl anionic group (probably an aspartic acid side-chain) in the vicinity of the hydrophobic core. A preliminary model is proposed for the overall folding of the kringle polypeptide chain.