Zygmunt Gryczynski

Functionally Different Agonists Induce Distinct Conformations in the G Protein Coupling Domain of the β2Adrenergic Receptor

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the β2 adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the β2 adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

2 – Basics of Fluorescence and FRET

This chapter discusses the most important characteristics of fluorescence that plays a fundamental role in understanding the basics and the applications of Forster (fluorescence) resonance (radiationless) energy transfer (FRET). FRET is the transfer of electronic excitation energy between isolated donor D and acceptor A of suitable spectroscopic properties. The donor molecules, typically, emit at shorter wavelengths, which overlap with the absorption spectrum of the acceptor. This energy transfer occurs without the appearance of the photon and is the result of long-range interactions between the D and A dipoles. The most important factors affecting FRET are the overlap integral, the quantum yield of the donor in the absence of the acceptor, and the orientation factor. The quantitative analysis of steady-state and time-resolved FRET measurements provides information on global structures and conformational dynamics, and reveals thermodynamic parameters for conformational transition. This information is essential for the understanding of biological functions of proteins, DNA/RNA, and other biological assemblies that are frequently mediated by transitions between alternative conformations.

Multi-pulse based approach to study dynamics of molecular assemblies with subwavelength resolution (Conference Presentation)

The long standing unmet need of optical microscopy has been imaging subcellular structures with nanometer precision with speed that will allow following physiological processes in real time. Herein we presenting a new approach (multi-pulse pumping with time-gated detection; MPP-TGD) to increase image resolution and most importantly to significantly improve imaging speed. Alternative change from single pulse to multiple-pulse excitation within continuous excitation trace (in interleave excitation mode) allows for the instantaneous and specific increase (many-folds) in the intensity of subwavelength sized object labeled with long-lived probes. This permits for quick localization of the object. Such intensity change (blinking) on demand can be done with MHz frequency allowing for ultrafast point localization several hundred folds faster than localization based on single molecule blinking. Much higher speed for super-resolution imaging will pave the way for obtaining real time functional information and probing structural rearrangements at the nanometer scale in-vitro and in-vivo. This will have a critical impact on many biomedical applications and enhance our understanding of many cellular functions.nWe use the microtubules as a model biological system with our new approach to studying microtubule dynamics in real time. The recent work based on single molecule localization microscopy (SMLM) (Mikhaylova et al., 2015) clearly indicates that microtubules are ~25 nm diameter hollow biopolymers that are organized in a closely spaced (about 20-70 nm apart) microtubule bundles. These structures are organized differently between axons and dendrites and their precise organization in different cell compartments is not completely understood.