OPTICAL TWEEZERS
People
Ciro Cecconi
Protein folding remains a major unsolved challenge for modern biophysics. Despite the experimental and theoretical efforts of many laboratories in the last 40 years, our understanding of the basic rules that govern the attainment of a protein structure is still incomplete. This lack of information is partly due to the inadequacy of conventional bulk methods to study a process that is highly heterogeneous. During folding, individual molecules are thought to follow different pathways and populate different intermediate structures on their journey to the native state. The experimental characterization of such a multitude of folding routes requires measurements at single molecule level, as ensemble measurements provide only average information.
Recently, a method has been developed to study the folding process of individual proteins using optical tweezers [1,2,3]. In these studies, single molecules are directly manipulated and their behavior under tension is described under the effect of a microscopically well-defined perturbation.
This method presents a number of advantages over more traditional bulk techniques, allowing one, among other things: i) to detect and characterize alternative, less probable folding trajectories, ii) to monitor in real-time fluctuations between different molecular conformations, iii) to measure the magnitude of the forces that hold together the protein's secondary and tertiary structures, iv) to measure directly the potential of mean-force of a molecule as a function of its extension, and v) to probe alternative regions of the energy landscape to those explored in experiments with thermal or chemical denaturation.
At the CNR-Istituto Nanoscienze S3 in Modena we have built a force-sensor dual-beam optical tweezers setup that operates by direct measurement of light momentum. This instrument allows for force-ramp, force-constant and force-jump experiments in different buffer conditions. We are currently using this setup to study the (un)folding processes of various proteins, including: Acyl-CoA Binding Protein, Neuronal Calcium Sensor-1 and HIV1 protease.
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Fig 1. Sketch of the experimental setup
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Publications
Direct Observation of the Three-State Folding of a Single Protein Molecule
C. Cecconi, E. Shank, C. Bustamante, and S. Marqusee
Science 309, 2057-2060 (2005).
Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers
C. Cecconi, E. Shank, S. Marqusee, and C. Bustamante
European Biophysics Journal 37, 729-738 (2008).
The folding cooperativity of a protein is controlled by the topology of its polypeptide chain
E. Shank, C. Cecconi, J. Dill, S. Marqusee, and C. Bustamante
Nature 465, 637-641 (2010).