Single molecule detection and spectroscopy opens a whole new range of applications to life sciences. A biomolecule, labeled with a single fluorescent dye, can be detected and its position can be tracked with high spatial resolution even in living cells. In contrast to ensemble measurements single molecule studies reveal the entire distribution of states. For example, if a molecule can exist in multiple conformational states, an ensemble study would yield an 'average state' which might not represent a true state at all. On the other hand, the true distribution of molecular conformations can be derived by measuring the molecules sequentially one-by-one.
Many biological processes are intiated and controlled by structural changes within macromolecular systems. For example, proteins are responsible for a multitude of biological functions, such as chemical catalysis, transport, regulation and movement. All functions are achieved based on the unique three-dimensional structure and dynamic properties of proteins. In order to study the underlying reaction mechanisms, a technique to visualize the protein structure, e.g distance and relative orientation of particular units within the protein, on a nanometer-scale is necessary.
Labeling the protein with two fluorescent dyes, one acting as an energy donor (D), the other as an acceptor (A), the energy transfer (fluorescence resonance energy transfer, FRET) between the two dyes can be used as a measure for the distance. For small distances, as shown in the left part of the scheme below, the transfer is efficient, thus leading to fluorescence from A and reducing the fluorescence of D. For increasing distances, the signal from D rises at the expense of the signal from A. The ratio of fluorescence intensities of A and D is a direct measure for the distance between the two dyes.

Role of Membrane Transport Proteins

The lipids which make up a cell membrane act as a barrier that prohibits certain molecules from crossing from one side of the cell to the other. Membrane proteins take on the role of ferrying molecules (ions, sugars, nucleosides, drugs) across a cell's membrane. The study of single transport proteins (transporters) is important because these proteins are flexible and can assume different conformational states. In fact, it is believed that the specific function of transporters is triggered by a sequence of conformational transitions. To reveal the different conformational states it is necessary to study one protein at a time. By using single-pair FRET it is possible to measure how the different parts of a protein move relative to each other, and a detailed protein structure and protein function relationship may be developed. Similar measurements may be conducted on proteins with mutations including those found in human disease to see how they structurally affect the proteins we study.

(A) Fluorescence image of immobilized GlpT proteins. Each bright spot represents the fluorescence of a donor molecule attached to a single GlpT protein. (B) Control experiment in which one element in the attachment strategy was left out. (C) Immobilization scheme (see text for details).

The single protein is tethered to a glass coverslip using our immobilization protocol schematically shown in figure C. Then the sample is raster-scanned in the focal plane of a tightly focused laser beam. The fluorescent dyes attached to the individual proteins emit light as they pass through the laser spot and the emitted light is collected and sent to either a single-photon detector or a CCD. Figure A shows the fluorescence from single proteins when the full attachment chain is in place. On the other hand, figure B shows that protein binding is not effective when the immobilization protocol is not followed. So far, our immobilization scheme has been shown to work for GlpT (a bacterial membrane protein) and cdAE1 a soluble human protein (cytoplasmic domain of AE1).

One of the proteins we are studying is cdAE1. It is the cytoplasmic domain of the human transmembrane protein AE1 found in red blood cells. cdAE1 serves as a model protein because its crystal structure is known. We use Cy3 maleimide as donor and Cy5.5 maleimide as acceptor. For this FRET pair we calculate a Foerster radius in water of Ro = 4.73 nm. This is the distance for which the energy transfer efficiency is 50%. Donor and acceptor molecules are bound to specific aminoacids (cysteins) of the protein's aminoacid sequence. While the specific binding sites are known the three-dimensional structure of the folded protein as well as the conformational changes associated with its function are not known. Thus, measuring the energy transfer efficiency between a FRET pair attached to a single protein allows us to construct or verify different protein models.

Histogram of distances measured between FRET pairs attached to single cdAE1 proteins. The histogram reveals two peaks indicating that the protein resides in two major conformations.

Ensemble titration experiments from LRET (Luminescence Resonance Energy Transfer) studies appear to show a two state system for the 201C mutant with a low-form 201C distance of R = 3.4-3.5 nm and a high-form 201C distance of R = 4.5 nm. Our spFRET results are in agreement with the predictions of this ensemble measurement. The current model assumes that an equilibrium forms between the low-form distance (L) and the high-form distance (H) and that lowering pH should decrease the population in the H form and increase the population in the L form. We see this behavior in the single molecule data even though the average distance values for both curves are similar. This data also shows that our attachment method does not significantly perturb the structure of cdAE1.

Acknowledgements:
This work is performed in collaboration with Dr. Phil Knauf’s Lab at the University of Rochester Medical Center. We would like to thank Dr. Da-Neng Wang, NYU, for the purified GlpT protein, and Dr. Philip Low, Purdue University, for the 201C plasmid.