Analysis of heterogeneous fluorescence decays in proteins. Using fluorescence lifetime of 8-anilino-1-naphthalenesulfonate to probe apomyoglobin unfolding at equilibrium

Abstract

The solvatochromic fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is one of the popular probes of protein folding. Folding kinetics is tracked with ANS fluorescence intensity, usually interpreted as a reflection of protein structure—the hydrophobicity of the binding environments. Such simplistic view overlooks the complicated nature of ANS–protein complexes: the fluorescence characteristics are convoluted results of the ground state populational distribution of the probe–protein complex, the structural changes in the protein and the excited state photophysics of the probe. Understanding of the interplay of these aspects is crucial in accurate interpretation of the protein dynamics. In this work, the fluorescence decay of ANS complexed with apomyoglobin at different conformations denatured by pH is modeled. The fluorescence decay of the ANS–apomyoglobin complex contains information on not only apomyoglobin structure but also molecular populational distributions. The challenge in modeling fluorescence decay profiles originates from the convolution of heterogeneous binding and excited-state relaxation of the fluorescent probe. We analyzed frequency-domain fluorescence lifetime data of ANS-apomyoglobin with both maximum entropy methods (MEM) and nonlinear least squares methods (NLLS). MEM recovers a model of two expanding-and-merging lifetime distributions for ANS–apomyoglobin in the equilibrium transition from the native (N) through an intermediate (I-1) to the acid-unfolded state U A. At pH 6.5 and above, when apomyoglobin is mostly populated at the N-state, ANS–apomyoglobin emits a predominant long-lifetime fluorescence from a relaxed charge transfer state S 1,CT of ANS, and a short-lifetime fluorescence that is mainly from a nascent excited-state S 1,np of ANS stabilized by the strong ANS–apomyoglobin interaction. Lowering the pH diminishes the contribution from the S 1,np state. Meanwhile, more protein molecules become populated at the U A state, which exhibits a short lifetime that is not distinguishable from the S 1,np state. At pH 3.4, when the population of the U A becomes significant, the short-lifetime fluorescence comes predominantly from ANS binding to the U A. Further lowering the pH leads to more exposure of the bound ANS. The long lifetime shifts toward and finally merges with the short lifetime and becomes one broad distribution that stands for ANS binding to the U A below pH 2.4. The above expanding-and-merging model is consistent with F-statistic analysis of NLLS models. The consistency of this model with the knowledge from the literature, as well as the continuity of the decay parameters changing upon experimental conditions are also crucial in drawing the conclusions.