Talaga Group
Description
and protein conformational dynamics
732-445-2192
Research Results - Method
Single molecule Methods development
We are developing new approaches to single molecule measurements to enhance our ability to extract useful information from them. Innovations include data acquisition and instrument automation, incorporation of microfluidics, development of new labeling methods, and investigation of various immobilization schemes.
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.
Direct Determination of Kinetic Rates from Single-Molecule Photon Arrival Trajectories Using Hidden Markov Models
The measurement of fluorescence from single protein molecules has become an important new tool in the study of dynamic processes, allowing for the direct visualization of the motions experienced by individual proteins and macromolecular complexes. The data from such single-molecule experiments are in the form of photon trajectories, consisting of arrival times and wavelength information on individual photons. The analysis of photon trajectories can be difficult, particularly if the motions are occurring at rates comparable to the photon arrival rate or in the presence of noise. In this paper, we introduce the use of hidden Markov models (HMMs) for the analysis of photon trajectory data that operate using the photon data directly, without the need for ensemble averaging of the data as implied by correlation function analysis. Using a simple kinetic model, we examine the relationship between the uncertainty in the estimates of the motional rate and the photon detection rate. Remarkably, we obtain relative uncertainties in the rate constants of as little as 3% even when the interconversion rate is equal to the photon detection rate, and the uncertainty increases to only 10% when the interconversion rate is 10 times the photon detection rate. This suggests that useful information can be obtained for much faster kinetic regimes than have typically been studied. We also examine the impact of background photons on the determination of the rate and demonstrate that the HMM-based approach is robust, displaying small uncertainties for background photon arrival rates approaching that of the signal. These results not only are relevant in establishing the theoretical limits on precision, but are also useful in the context of experimental design. Finally, to demonstrate how the methodology can be extended to more complex kinetic models and how it can allow one to make use of the full power of statistics for purposes of model evaluation and selection, we consider a four-state kinetic model for protein conformational transitions previously studied by Schenter et al. (J. Phys. Chem. A 1999, 103, 10477). We show how an HMM can be used as an alternative to higher-order correlation function analysis for the detection of “conformational memory” and apparent non-Markovian dynamics arising from such temporally inhomogeneous kinetic schemes.
Production of a microfluidic mixer in PDMS
This multi-channel sorting mixer was produced from a negative mold of photoresist on silicon. The mold creates an embedded design in PDMS. Subsequent bonding of the PDMS to a glass slide makes a liquid-tight seal. These fluidic devices are being used to confine bio-molecules to an observable volume by confocal microscopy so that we may watch the bio-molecules as they perform their function in real time.
Chromophore counting by discrete photobleaching events.
Development of micro/nano fluidic devices for Amyloid Detection
June 22, 2005
Hidden Markov Models for Diffusive Processes
The top panel (a) shows a Monte Carlo simulated trajectory of a molecule diffusing through a spherically symmetric Gaussian collection volume. The next panel (b) shows the reciprocal of the inter-photon time. The delay between excitation and emission of the photon is the lifetime and is shown in panel (c). Panels (b) and (c) are used with three dimensional diffusion of the molecule through the focus of the instrument as the hidden Markov model to reconstruct the Brownian motion trajectory in panel (d). The trajectory was reconstructed by using Monte Carlo sampling of the HMM parameters to maximize the likelihood of the data in (b) and (c).
Single particle tracking of albumin nano-particles.
In collaboration with the group of Prabhas Moghe we are conducting studies to follow the trafficking of single albumin nano-particles that have been functionalized with ligands to modulate cellular motility.
The probability-normalized (∑P(O|S)=1) fluorescence spectra of C153 in hexane (blue) and in methanol (green). The mutual information in bits between the state (polar versus nonpolar) and each photon emitted (red).Information theory for 2 color single molecule experiments
From an information theory point of view the molecule encodes information into photons using the dyes as a transducer. The photons are converted into raw data by the detection apparatus and then decoded into a useful form by some data analysis procedure. From the reduced data we draw inferences about the molecule based on the data and our prior knowledge of the system. Two color experiments represent one of the most common types of single molecule fluorescence measurements. We developed a formulation of Shannons information theory to treat two-color problems and showed how it can be used to design experiments based on the number of photons required to deliver a particular amount of information.
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.
Markov processes in single molecule fluorescence
David S. Talaga, Current Opinion in Colloid & Interface Science 12:6 (2007) 285-296
This article examines the current status of Markov processes in single molecule fluorescence. For molecular dynamics to be described by a Markov process, the Markov process must include all states involved in the dynamics and the FPT distributions out of those states must be describable by a simple exponential law. The observation of non-exponential first-passage time distributions or other evidence of non-Markovian dynamics is common in single molecule studies and offers an opportunity to expand the Markov model to include new dynamics or states that improve understanding of the system.
Link to published version