Conformation of exciton-coupled molecular dimers by phase-modulation 2D fluorescence spectroscopy |
|
| Andrew Marcus | |
| University of Oregon, Eugene | |
| The ability to determine three-dimensional structures of macromolecules and macromolecular complexes plays a central role in the fields of molecular biology and material science. Methods to extract structural information from experimental observations such as X-ray crystallography, NMR, and optical spectroscopy are routinely applied to a diverse array of problems, ranging from investigations of biological structure-function relationships to the chemical basis of molecular recognition. In recent years, two-dimensional (2D) optical methods have become well established to reveal incisive information about non-crystalline macromolecular systems - information that is not readily obtainable by conventional linear spectroscopic techniques. 2D optical spectroscopy probes the nanometer-scale couplings between vibrational or electronic transition dipole moments of neighboring chemical groups, similar to the way NMR detects the angstrom-scale couplings between adjacent nuclear spins in molecules. Following the examples established by 2D Fourier transform spectroscopies, 2D electronic spectroscopy (2D ES) holds promise as a general approach for the structural analysis of non-crystalline macromolecular systems, albeit for the nanometer length scales over which electronic couplings occur. Here we demonstrate a phase-modulation approach to 2D ES that sensitively detects fluorescence to resolve the exciton coupling in dimers of magnesium meso tetraphenylporphyrin (MgTPP), which are embedded in 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) liposomal vesicles. MgTPP is a non-polar molecule that preferentially enters the low dielectric amphiphilic regions of the phospholipid bilayer. At intermediate concentration, MgTPP forms dimers as evidenced by changes in the linear and 2D absorption spectra. Quantitative comparison between our measurements and simulated spectra for a broad distribution of selected conformations, screened by a global optimization procedure, shows that the information contained in linear spectra alone is not sufficient to determine a unique structure. In contrast, the additional information provided by 2D spectra constrains a narrow distribution of “T-shaped” conformations, which are specified by the relative separation and orientations of the MgTPP macrocycles. In our approach, called phase-modulation 2D fluorescence spectroscopy (PM-2D FS), a collinear sequence of four laser pulses is used to excite electronic population. The ensuing nonlinear signal is detected by sweeping the relative phases of the excitation pulses at kHz frequencies, and by using lock-in amplification to monitor the spontaneous fluorescence. This technique enables phase-selective detection of fluorescence at sufficiently high frequencies to effectively reduce laboratory 1/f noise. Because the PM-2D FS observable depends on nonlinear populations that generate fluorescence, a different combination of nonlinear coherence terms contribute to the signal than those of standard photon-echo 2D ES. The PM-2D FS method might be widely applied to problems of biological and material significance. Spectroscopic studies of macromolecular conformation, based on exciton-coupled labels could be practically employed to extract detailed structural information. PM-2D FS opens new possibilities to study exciton-coupling under low light conditions, in part due to its high sensitivity. This feature may facilitate future 2D experiments on single molecules, and on UV-absorbing chromophores. |