J. Phys. Chem. B 2010, 114, 12383–12391
12383
Resolving Inhomogeneity Using Lifetime-Weighted Fluorescence Correlation Spectroscopy Kunihiko Ishii and Tahei Tahara* Molecular Spectroscopy Laboratory, AdVanced Science Institute (ASI), RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan ReceiVed: May 10, 2010; ReVised Manuscript ReceiVed: July 14, 2010
Fluorescence correlation spectroscopy (FCS) was extended by incorporating information of the fluorescence lifetime. This new experimental approach, called lifetime-weighted FCS, enables us to observe fluorescence lifetime fluctuations in the nano- to millisecond time region. The potential of this method for resolving inhomogeneity in complex systems was demonstrated. First, by measuring a mixture of two dye molecules having different fluorescence lifetimes, it was shown that the lifetime-weighted correlation deviates from the ordinary intensity correlation only when the system is inhomogeneous. This demonstrated that lifetime-weighted FCS is capable of detecting inhomogeneity in an ensemble-averaged fluorescence decay profile without any a priori knowledge about the system. Second, we applied this method to a dye-labeled polypeptide, a prototypical model of complex biopolymers. It was found that the ratio between the lifetime-weighted and ordinary intensity correlation changes with change of the environment around the polypeptide. This result was interpreted in terms of environment-dependent conformational inhomogeneity of the polypeptide. Delay time dependence of the ratio was found to be constant from ∼1 µs to several milliseconds, indicating that the observed inhomogeneity is persistent in the measured time scale. In combination with fluorescence intensity correlation, lifetime-weighted FCS allows us to examine conformational fluctuations of complex systems in the time region from nano- to milliseconds, being free from the translational diffusion signal. 1. Introduction Complex macromolecules such as biopolymers have a large number of conformations in solution, and their interconversion takes place in a wide range of characteristic time scales. To uncover such conformational distribution and structural fluctuation, fluorescence lifetime measurements of an appropriately attached fluorophore are widely utilized. The fluorescence lifetime is an excellent probe for the conformational difference of the macromolecules because fluorescence quenching effects such as FRET (Fo¨rster resonance energy transfer) are very sensitive to the change of the conformation.1 Therefore, conformational inhomogeneity of the macromolecules can be investigated by examining the fluorescence lifetime distribution of the appropriate fluorophores. This is an issue of current interest because the flexibility of complex molecules plays crucial roles in many biological functions and is an essential part of the physical chemistry of macromolecules. We can extract information about the lifetime distribution (or the lifetime inhomogeneity) from ensemble-averaged fluorescence decay data using the multiexponential fitting analysis or, more elaborately, by the maximum entropy method (MEM).2-5 Especially, MEM enables a model-free estimate of the lifetime distribution in the fluorescence decay observed. However, these analyses cannot distinguish such conformational inhomogeneity from an intrinsic nonexponential dynamics of the fluorophore because the data themselves are an average over a large number of molecules. In fact, a multiexponential fluorescence decay does not necessarily imply the existence of multiple species because it may arise from the contribution of the highly excited states or vibrational and solvent relaxations. More importantly, ensemble-averaged data are completely insensitive to the * To whom correspondence should be addressed. Fax: +81-48-467-4539. E-mail:
[email protected].
dynamical aspect of inhomogeneity, for example, interconversion rates between different conformers. Therefore, we need a molecule-by-molecule approach6 to directly resolve the inhomogeneity of the fluorescence lifetime and examine the temporal change of the fluorescence lifetime (lifetime fluctuation) that arises from conformational interconversion. Several groups have studied the fluorescence lifetime fluctuation of single molecules by measuring averaged fluorescence lifetimes in millisecond time windows using the time-correlated single-photon counting (TCSPC) method.7-11 Although this approach is straightforward and powerful, we cannot approach a time region shorter than a millisecond because a moderate number of photons (typically ∼100) is required to determine the fluorescence lifetime. For the shorter time region (
