Characterization of Local Polarity and Hydrophobic Binding Sites of β-Lactoglobulin by Using N-Terminal Specific Fluorescence Labeling Su-Ying Dong, Zhen-Wen Zhao, and Hui-Min Ma* Center for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China Received June 29, 2005
Because of wide ligand-binding ability and significant industrial interest of β-lactoglobulin (β-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 β-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 β-LG have been characterized by using N-terminal specific fluorescence labeling combined with a polarity-sensitive fluorescent probe 3-(4-chloro-6-hydrazino- 1,3,5triazinylamino)-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 β-LG is decreased after thermal denaturation. This polarity trend toward decreasing instead of increasing shows that β-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 β-LG is found to be relevant to not only experimental conditions (pH e 6.2 or pH > 7.1) but also binding mechanisms (hydrophobic affinity or electrostatic interaction). Keywords: characterization of hydrophobic binding sites • local polarity detection • N-terminal specific labeling • β-lactoglobulin
Introduction β-Lactoglobulin (β-LG), a member of the lipocalin family, is the most abundant protein in the whey (almost 50% of the total protein) of bovine milk, whose property tends to determine the behavior of the total whey protein system.1-5 The structure of β-LG has been well-characterized.2,3 The protein is a small globular one with a monomer molecular weight of 18.3 kDa6,7 and contains 162 amino acid residues,1,2,4-6 among which 34% of the residues are nonpolar.5 β-LG with a leucine residue as N-terminus has a β-barrel (calyx), formed by 8 antiparallel β-strands (A-H), and a 3-turn R-helix attached to the outside of the barrel, followed by an extra β-strand (I).1 Through this extra strand, β-LG usually associates into a homodimer but can dissociate into monomers at neutral pH and low concentrations (550 nm that can avoid the interferences of the background fluorescence shorter than 500 nm from common proteins, and more importantly, its maximum emission wavelength (λem) is sensitive only to the surrounding polarity but not to pH and temperature. Furthermore, the transamination reaction leads to a single site-specific labeling of the probe on the N-terminal amino group without modification of the internal amino groups on lysine residues,23,24 thus allowing the accurate determination of local polarity of N-terminal domain. We envisioned that this method of local polarity detection may be useful in exploring the binding behavior of β-LG. Specifically, in the present study we take advantages of both the probe’s property and the sitespecific feature of N-terminal labeling, to determine the local polarity change of β-LG during thermal denaturation and thereby to characterize its ligand-binding properties. By measuring and comparing the local polarity changes of the two systems of CHTDP-labeled β-LG, and the simple mixture of β-LG and CHTDP, the first quantitative information on the polarities of the N-terminal domain and the internal binding site in β-LG has been obtained, and it is revealed that β-LG may have no fixed external hydrophobic binding site. On the basis of this finding, together with previous binding studies, it is believed that the location of ligand binding in β-LG is associated with not only experimental conditions (pH e 6.2 or pH > 7.1) but also binding modes (hydrophobic affinity or electrostatic interaction).
Experimental Section Reagents. Bovine β-LG (CAT No. 151536, ICN Biomedicals, Inc., USA), glyoxylic acid (Acros), and sodium cyanoborohydride (Sigma) were used as received. A 4 mM solution of CHTDP, synthesized as reported previously,23 was prepared in dimethyl sulfoxide (DMSO). Protein solutions (
