Characterization of local polarity and hydrophobic binding sites of beta-lactoglobulin by using N-terminal specific fluorescence labeling.
Because of wide ligand-binding ability and significant industrial interest of beta-lactoglobulin (beta-LG), its binding properties have been extensively studied. However, there still exists a controversy as to where a ligand binds, since at least two potential hydrophobic binding sites in beta-LG have been postulated for ligand binding: an internal one (calyx) and an external one (near the N-terminus). In this work, the local polarity and hydrophobic binding sites of beta-LG have been characterized by using N-terminal specific fluorescence labeling combined with a polarity-sensitive fluorescent probe 3-(4-chloro-6-hydrazino- 1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CHTDP). The polarity within the calyx is found to be extremely low, which is explained in terms of superhydrophobicity possibly resulting from its nanostructure, and the polarity is increased with the destruction of the calyx by heat treatment. However, the polarity of the N-terminal domain in native beta-LG is decreased after thermal denaturation. This polarity trend toward decreasing instead of increasing shows that beta-LG may have no definite external hydrophobic binding site. The hydrophobic binding of a ligand such as CHTDP at the surface of the protein is probably achieved via appropriate assembling of corresponding hydrophobic residues rather than via a fixed external hydrophobic binding site. Also, the ligand-binding location in beta-LG is found to be relevant to not only experimental conditions (pH < or = 6.2 or pH > 7.1) but also binding mechanisms (hydrophobic affinity or electrostatic interaction).
Dong
Su-Ying
SY
Center for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China.
Zhao
Zhen-Wen
ZW
Ma
Hui-Min
HM
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry; D057927:Hydrophobic and Hydrophilic Interactions; D007782:Lactoglobulins / Q000737:chemistry*; D010619:Phenazines / Q000737:chemistry; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary
C508513:3-(4-chloro-6-hydrazino-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine; D005456:Fluorescent Dyes; D007782:Lactoglobulins; D010619:Phenazines