Single-molecule spectroscopic techniques were applied to individual pigments embedded in a chromoprotein. A sensitive tool to monitor structural fluctuations of the protein backbone in the local environment of the chromophore is provided by recording the changes of the spectral positions of the pigment absorptions as a function of time. The data provide information about the organization of the energy landscape of the protein in tiers that can be characterized by an average barrier height. Additionally, a correlation between the average barrier height within a distinct tier and the time scale of the structural fluctuations is observed. P roteins are supramolecular machines that perform a tremendous variety of tasks in living organisms, such as the transport of electrons and small molecules, catalysis of biochemical reactions, or storage of energy to fuel metabolic processes. Despite the multitude of functions associated with proteins, they all consist of a linear chain of covalently linked amino acids. The high specificity of a particular protein results from its complex three-dimensional structure. The primary polypeptide sequence folds into secondary structural elements, such as ␣-helices and ␤-sheets, and the secondary structures are folded into a compact three-dimensional tertiary arrangement that determines the biological role and the status of activity of the protein. Proteins are remarkably robust despite the fact that their structure is stabilized only by relatively weak peptide-peptide and proteinsolvent interactions, such as hydrogen bonds and hydrophobic interactions. The connection among protein folding, protein structure, and protein function is one of the greatest challenges of current research. A major question that arises is: how does a protein fold within a reasonable time into its biologically active form?Even for a small protein consisting of Ϸ100 amino acids, the number of possible conformations is Ϸ10 100. Because of the weak interactions that stabilize the protein and the many degrees of freedom of such a large molecule, the lowest energy state is not unique, and description in terms of a rugged energy landscape is appropriate. The term ''energy landscape'' refers to the potential energy hypersurface of Ϸ 3,000 dimensions, resulting from the coordinates of the atoms of the protein. It features a large number of minima, maxima, and saddle points, and each minimum in this landscape represents a different conformational substate (CS) that corresponds to a different arrangement of the atoms. However, the number of possible conformations of a protein is so large that folding into the native state within a reasonable time by a process of statistical trial and error is impossible (Levinthal's paradox). The contemporary picture is that protein folding occurs by a progressive stabilization of intermediates that retains partially ''correct'' folding units guided by interactions that stabilize subdomains and domains of the final folded state.To describe protein dynamics and function, a model has been ...

We study single dibenzoterrylene molecules in an anthracene single crystal at 1.4 K in two insertion sites at 785.1 and 794.3 nm. The single-molecule zero-phonon lines are narrow (about 30 MHz), intense (the detected fluorescence rates at saturation reach 100,000 counts s(-1)), and very photostable. The intersystem-crossing yield is extremely low (10(-7) or lower). All of these features are hallmarks of an excellent system for high-resolution spectroscopy and nanoscale probing at cryogenic temperatures.

We present a spectroscopic study of the properties of the two principal insertion sites (at 785.1 and 794.3 nm) of single dibenzoterrylene molecules in anthracene single crystals at cryogenic temperatures. We measured the temperature dependence of the line width, the orientation of the transition dipole moments, and the Stark effect. We performed molecular dynamics simulations, which show that one dibenzoterrylene molecule preferably replaces three anthracene molecules. From simulated annealing, we derive the molecular conformations in the most stable insertion sites and the orientations of the transition dipole moments. The good agreement between the spectroscopic results and the simulations allows us to propose unambiguous structures for the two principal spectroscopic sites.

The full exploitation of single-molecule spectroscopy in disordered systems is often hampered by spectral diffusion processes of the optical transitions due to structural fluctuations in the local environment of the probe molecule which leads to temporal averaging of the signal. Multivariate statistical pattern recognition techniques, originally developed for single-molecule cryoelectron microscopy, allow us to retrieve detailed information from optical single-molecule spectra. As an example, we present the phonon side band of the B800 excitations of the light-harvesting 2 (LH2) complex from Rhodospirillum molischianum, revealing the electron-phonon coupling strength for these transitions. The measured Debye-Waller factors, ranging from 0.4 to 0.9, fall in the regime of weak electron-phonon coupling.