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Zhuang X. Science 288, 2048 (2000).
Keywords
SINGLE MOLECULE, GREEN FLUORESCENT PROTEIN, CONFORMATIONAL SUBSTATES, PROTEIN FUNCTIONAL DYNAMICS, FOLDING AND UNFOLDING, FREE ENERGY CONFIGURATION LANDSCAPE, SILICA GEL ENCAPSULATION, FLUORESCENCE, CIRCULAR DICHROISM

SINGLE MOLECULE INVESTIGATIONS OF DISCRETE SUBSTATES AND FOLDING-UNFOLDING PATHWAYS OF GREEN FLUORESCENT PROTEIN: EXPERIMENT AND THEORY

Università degli Studi di Milano-Bicocca
Abstract
Proteins have a chemical composition, number and types of amminoacids, that determines their folded three-dimensional structures. They can undergo a variety of (fast) vibrations and (slower) structural rearrangements in multi-dimensional energy landscape characterized by a multitude of almost iso-energetic minima. Each of these minima corresponds to one particular protein `conformational substate'. Among proteins, the Green Fluorescent Protein (GFP) is an ideal candidate for detailed conformational investigations, since it is naturally fluorescent, and its chromophore is sensitive to its environment.
The program is devoted to the detection of the predicted multiplicity of native conformational states of GFP mutants. Evidence of substates, although long expected, has been very hard to prove with ensemble measurements since the latter yield average values of the individual properties. Recent progress in single molecule spectroscopy makes this goal possible nowadays. In particular single molecule fluorescence spectroscopy on immobilized GFP mutants will be employed here with the theoretical contribution of numerical simulations. This approach is based on the close collaboration of three research groups with proven competence in complementary scientific areas: One and two photon excitation single molecule fluorescence spectroscopy at high space and time resolution (MIB); Expression, purification and silica gel encapsulation of GFP mutants and their bulk >>>

Principal Investigator
Giancarlo Baldini Università degli Studi di MILANO-BICOCCA
Research Objectives
The main objective of the program is the Detection/characterization of the Green Fluorescent Protein (GFP) native substates and of their evolution during unfolding. This will be achieved by single molecule fluorescence spectroscopy on GFP mutants trapped in wet gels and described by numerical simulations of protein internal dynamics.

Specifically the objectives are:

A) Experimental spectroscopic investigation at the single molecule level (Milano-Bicocca,MIB) of the native substates of GFPmut2 molecules encapsulated in wet silica gel (Parma,PR). This mutant is particularly bright and efficiently produced. Two of the units (MIB and PR) have thoroughly characterized the fluorescence dynamics of this protein, in terms of fluorescence blinking and switching between the blue and green emission peculiar of the neutral and anionic state of the chromophore. We propose here to take the anionic (A) to neutral (N) switching rate as the key parameter to be monitored in order to investigate the protein substates and dynamics (“protein motion”). This choice is based on previous published results of the MIB and PR units.
B) Parallel predictions and comparisons with simulations (SISSA) on this protein mutant. The SISSA unit will perform theoretical/simulative topological characterization of substates transition in GFP. A novel Gaussian framework, recently introduced and tested by the SISSA group, will be applied in the context of GFP. By this low-level >>>

Timescale
24 months
National and international background
Generalities on the folding /unfolding mechanism

Proteins are polypeptides which consist, typically, of hundreds to thousands amino acids and fold into protein-specific three-dimensional structures In the past decade there has been a remarkable increase in the number of studies where the kinetics and thermodynamics of proteins is investigated by means of tools and concepts borrowed from statistical mechanics and condensed matter physics. The reason for the interest of the biophysical community for biopolymers largely resides in the unique properties that the latter possess by comparison to random heteropolymers. The most fundamental of these differences is constituted by the capability of proteins to fold rapidly into the native state, the conformation with maximum biological activity (Daura 1997). The so called native structure is determined by the specific amino acid sequence of the protein, which is genetically encoded. Proteins have not rigid structures, however, and can undergo a variety of (fast) vibrations and (slower) structural rearrangements, the latter being called 'conformational transitions'. This situation can be summarized by a one-dimensional sketch of the complex, multi-dimensional (~104 coordinates) energy landscape, which determines the correspondingly complex dynamics of a protein, as originally suggested by Hans Frauenfelder (Frauenfelder 1988, Frauenfelder 1991, Frauenfelder 2001). This energy landscape is characterized by a >>>