Research Results - Folding

Single molecule protein/peptide conformational dynamics

We are interested in understanding the mechanisms of protein and peptide conformational dynamics including structural changes that occur upon ligand binding as well as protein folding and misfolding.

During their life cycle proteins undergo many types ofconformational changes. After folding, the activity and regulation of proteins are also mediated by conformational changes. Misfolded and damaged proteins are usually degraded. However, irreversible aggregation into amyloid is accompanied by yet another conformational change.

Protein Conformational Dynamics Background

During their life cycle proteins undergo many types of conformational changes.  The first type of conformational change is the process of protein folding.  The polypeptide must fold into its active three-dimensional structure before it is degraded by cellular proteases or aggregates. In fact this process is so important that organisms have developed systems such as Chaperonins that consume energy to assist in the folding process. Once the protein has obtained its proper three-dimensional structure it may undergo further structural changes that are associated with its function. Many enzymes and ligand-binding proteins must undergo large conformational changes during their function. The activity of a protein can be modulated by allosteric interactions with other molecules. These interactions result in a dynamic structural change that changes the protein's activity. Finally, most proteins only survive a short time in the cell without becoming damaged.  Damage often will change the structure of the protein and allow proteases to degrade it allowing the cycle to begin anew.  If the degradation process is faulty then aggregation can occur.  Thus, during their life cycle proteins can undergo five basic classes of conformational change.

  • Folding
  • Activity
  • Regulation
  • Degradation
  • Aggregation
Defects in these processes can result in diseases.

Pnas 2000 V97 P13021

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Single Molecule Dynamics Associated with Protein Folding and  Deformations of Light-Harvesting Complexes

Single Molecule Dynamics Associated with Protein Folding and Deformations of Light-Harvesting Complexes.

Two new applications of single molecule methods in biology are described. In one, single assemblies of the intact light harvesting complex LH2 from Rhodopseudomonas acidophila were bound to mica surfaces at 300K and examined by observing their fluorescence after polarized light excitation. They mostly behaved as electrically elliptic absorbers whose ellipticity fluctuates, showing that there is a mobile structural deformation. The other application involves the folding and unfolding of a coiled coil GCN4-P1 peptides. By following the trajectory of individual members of a folding ensemble we are able to evaluate and distributions of properties not available from bulk studies.

2Gbp Rendered-1

Conformational changes during ligand recognition in Glucose/Galactose Binding Protein

Glucose/Galactose Binding Protein (GBP) is a receptor in the chemosensory pathway of bacterial chemotaxis. GBP consists of two domains, each of which contains a beta-sheet packed between alpha-helices. The binding cleft is between the two hinged domains. Signal transduction begins in the periplasmic compartment where GBP is located. Binding of glucose or galatose by GBP causes a large amplitude conformational change that encapsulates the ligand. This allows GBP to bind to a transmembrane receptor initiating the remainder of the chemosensory pathway that regulates the bacterial flagellar motor and determines swimming behavior of the cell in response to chemical atractants or repellents

All 3 By Tmrc-1

Electron Transfer Probes of Peptide/Protein Backbone Conformation

Single molecule polyproline isomerization is studied by fluorescence quenching, induced by short-ranged electron transfer between TMR(5-carboxytetramethylrhodamine) and DMPD(dimethyl-p-phenylenediamine). To do this, we have prepared a polyproline(n=2,3) peptide with DMPD at its carboxylic end and TMR at its amino end. The electron transfer efficiency is measured by TCSPC(time-correlated single photon counting) in which accepter fluorescence lifetime is comparatively quenched according to the proximity of donor molecule. This allows us to measure local protein or peptide conformation at the level of a few residues. It also allows us to investigate the role of conformation in biological electron transfer. The figure above shows the major trans configuration on the left and the cis configuration on the right. The structures were determined by NMR NOE constraints. The trans configuration corresponds to the Pro-II helix and the cis configuration corresponds to the Pro-I helix.

Nanopore Amyloid

Solid-state nanopore measurements of amyloid formation

In collaboration with the group of Jiali Li at the University of Arkansas Department of Physics we have begun making solid state nanopore electrical measurements on the aggregates that form during assembly of amyloid from ß-lactoglobulin. Our preliminary assessment of the data suggests that the solid state nanopore can distinguish between different classes of amyloid species.

β-Lactoglobulin Calyx Dynamics

β-Lactoglobulin Calyx Dynamics
The formation of a hydrophobic core provides a large part of the driving force for protein folding.
We are investigating amyloid formation from β-lactoglobulin and are therefore interested in understanding how the dynamics of core stability are influenced by solution conditions.
We have exploited the presence of a large hydrophobic binding pocket in β-lactoglobulin to encapsulate coumarin 153 (C153).
Steady state spectroscopy reveals a very blue-shifted spectrum consistent with an environment similar to a combination of hexane and toluene.
Time resolved fluorescence Stokes shift measurements reflect the dynamics of the hydrophobic core of β-lactoglobulin.
A transition in the dynamics of the hydrophobic core occurs at a lower temperature than does the melting of the protein as measured by circular dichroism suggesting a partitioning of  the enthalpy and entropy balance between the core structure and secondary structure.

Messina2007

Protein Free Energy Landscapes Remodeled by Ligand Binding

Glucose/galactose binding protein (GGBP) functions in two different larger systems of proteins used by enteric bacteria for molecular recognition and signaling. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP. Three fluorescence components appear at zero glucose concentration and systematically transition to three compo- nents at high glucose concentration. Fluorescence anisotropy correlations, fluorescent lifetimes, thermodynamics, computational structure minimization, and literature work were used to assign the three components as open, closed, and twisted conformations of the protein. The existence of three states at all glucose concentrations indicates that the protein continuously fluctuates about its conformational state space via thermally driven state transitions; glucose biases the populations by reorganizing the free energy profile. These results and their implications are discussed in terms of the two types of specific and nonspecific interactions GGBP has with cytoplasmic membrane proteins.