Single Molecule Spectroscopy: Nobel Conference Lectures: R. Rigler, M. Orrit, and T. Basche, Editors Single Molecule Dynamics Associated with Protein Folding and Deformations of Light-Harvesting Complexes. David S. Talaga* ‡ , Yiwei Jia*, Martin A. Bopp*, Alexander Sytnik*, William A. DeGrado # , Richard J. Cogdell † and Robin M. Hochstrasser* ∗ *Chemistry Department and NIH Laser Resource, University of Pennsylvania, Philadelphia, PA, 19104, USA # Department of Biochemistry and Biophysics, University of Pennsylvania † Division of Biochemistry and Molecular Biology, Institute of Biomedi- cal and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK ‡ Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, New Brunswick/Piscataway, NJ 08854 † (Dated: February 5, 2002) We present two applications of single mol. spectroscopy that illustrate how ensemble averaging masks important dynamic properties of fluctuating systems. In the first, mobile elliptical struc- tural deformations are obsd. in single assemblies of the light harvesting complex, LH2. These mobile structural deformations are averaged in bulk measurements resulting in the erroneous conclusion that LHS is a circular absorber. In the second application, trajectories of individual members of a folding ensemble of coiled coil GCN4-P1 peptides allow us to determine distribu- tions of properties not available from bulk studies. Keywords: I. INTRODUCTION Experimental understanding of biological and chemical systems is based primarily on measure- ments of many molecules and therefore the evolution of the mean of that ensemble. However, since het- erogeneity of structure and mechanism is required to describe complex systems such as proteins and other biological assemblies, this useful paradigm can break down. Recent developments in single molecule de- tection have allowed the study of single molecules and single biological assemblies under physiological conditions. By following the trajectory of individual members of an equilibrium ensemble as they evolve in time fluctuation rates, reaction rate constants, and distributions of other properties can be evaluated. We present two applications of single molecule spectroscopy that illustrate how ensemble averaging masks important dynamic properties fluctuating sys- tems. In the first, mobile elliptical structural defor- mations are observed in single assemblies of the light harvesting complex, LH2. These mobile structural deformations are averaged in bulk measurements re- sulting in the erroneous conclusion that LH2 is a cir- cular absorber. In the second application trajectories of individual members of a folding ensemble of coiled coil GCN4-P1 peptides allow us to determine distri- butions of properties not available from bulk studies. ∗ Electronic address: hochstra@sas.upenn.edu, talaga@rutgers.edu † URL: http://talaga.rutgers.edu II. STRUCTURAL DEFORMATIONS OF SURFACE-IMMOBILIZED SINGLE LIGHT-HARVESTING COMPLEXES. 1 The crystal structure of the LH2 complex from pho- tosynthetic bacteria Rhodopseudomonas acidophila is notable for its high symmetry arrangement of the nine αβ-dipeptides which form the scaffold holding the associated bacteriochlorophyll (Bchl) cofactors. 2 The LH2 Bchls are arranged into two rings that have approximate 9-fold rotation symmetry. The B800 ring contains nine monomeric Bchls located between the β-apoproteins. The B850 ring consists of nine pairs of Bchls each associated with one αβ-dipeptide. In the LH2 complex the B800 ring absorbs light and transfers the excitation energy to B850 in less than a picosecond. 3 The excitation properties of macromolec- ular systems depend on the interplay between the nu- clear motions that tend to localize excitations, and the delocalizing effect of the interaction between the molecules. 4 Therefore the nature of the excitation and energy transfer in the LH2 complex must depend not only on the static or average structure but also on the structural fluctuations that can occur in bacterial membrane. Single molecule methods are well suited for the investigation of the microseconds to seconds structural dynamics. 5 Previously we 6 and others 7 ap- plied single molecule confocal microscopy for the pho- tophysical and photochemical characterization of the LH2 complexes. The LH2 complexes were immobilized on a mica surface to model the protein interactions occur- ring in bacterial cells. The mica has some nega- tive charges on its atomically flat surface but it can