France: Postdoc Offer Structure Function Relationships In Proteins Using Single Molecule Fluorescence Structure/Function Relationship in Proteins

Postdoc Offer Structure Function Relationships In Proteins Using Single Molecule Fluorescence
Structure/function relationship in proteins, France

We are looking for a post-doctoral candidate with proven expertise in fluorescent techniques applied to the study of single molecules (FRET, FCS, FIONA, etc.). The project is described in the following but essentially the idea is to study the activity of a single enzyme as a function of temperature and tension with the aim of understanding the relationship between the function of an enzyme and its structure (obtained from numerical simulations (done in collaboration with the group of M.Baaden) about the known structure of the investigated protein).

Interested candidates should send a CV, a letter of motivation and the email address of three referees to David Bensimon: david[ at ]lps.ens.fr

1-Motivation
Understanding the relation between the structure of a protein and its sequence remains one of the major problems of the post-genomic area in Biology. The difficulty of solving that problem is compounded by the existence of many possible metastable configurations in the protein folding pathway. Related to that problem is the question of the uniqueness of the native, ground-state of a protein. If the energy landscape of a protein is sufficiently complex and rugged (similar to that of a glass as is often claimed) then quite possibly the ground state of a protein could be degenerate or energetically close to other structural states. Indeed, recent experiments using FCS and other fluorescent methods (Xie et al., Rigler et al.) suggest that the ground state of a protein is not unique and that the protein may hop between structurally different states whereby its activity is affected. There is increasing evidence that this flexibility in the structure of a protein can be essential for its function: either by allowing for the deformation necessary for its catalytic activity (in the case of an enzyme) or by allowing for binding to its substrate through induced fit mechanisms.

2-Description of the project, methodology
The goal of this project is to study the relation between the structural fluctuations of an enzyme and its catalytic function. For this purpose we will use a dual theoretical/experimental approach. From a theoretical point of view, while predicting the structure of a protein from its sequence is a very difficult problem, studying the fluctuations of a protein near its crystallographic structure is much more feasible. We will therefore use the known structure of a number of enzymes to theoretically investigate the possible structural deformations of the protein near equilibrium. This will inform us about the soft deformation modes of the enzyme and the possible existence of nearby metastable states.
We will then use this information to compare and interpret a number of experiment aimed at investigating the functional fluctuations of an enzyme under various conditions of buffers, temperature, tension, etc. To monitor the enzymatic activity we will use enzymes that catalyse a reaction whose end product is fluorescent. Thus by monitoring the fluorescent out-bursts by single molecule techniques we will be able to monitor the enzymatic cycles one by one and study their correlation, catalytic rate, affinity, etc. as a function of time and of the various constraints imposed on the enzyme. The first constraint we will study is the temperature. We will embed enzymes in a gel and study the temperature dependence of their functional cycle. The second constraint we will impose is tension in the enzyme. This will be attempted along two routes. Either via a DNA segment tethered at two places on the enzyme (a technique recently introduced by G. Zocchi) or by anchoring the enzyme on a surface and pulling on it with a known force using a DNA tether bound to a magnetic bead and magnetic tweezers to pull on the bead.

3-Expected results :
We expect to see a correlation of enzymatic catalytic rates on a short time scale and a decorrelation on a longer time scale indicative of the existence of different functionally active states of the enzyme. We expect the number of these states and the correlation times to vary as the temperature or the tension on the enzyme is altered. Different mutants and enzymes from different sources (psychrotrophic, mesophilic and thermophilic bacteria) will be compared. These experimental data will be compared with numerical simulations and analysis of the enzyme’s fluctuations near its crystallographically known state both as a function of temperature and of the tension between known positions on the enzyme. This dual approach will help us understand how the activity of an enzyme is dependent on and modulated by its flexibility, fluctuations and possible manifold of metastable ground-states.

References :
Xie, S. N. (2001). “Single-molecule approach to enzymology.” Single Molecules 2(4): 229-236 ;
Rigler, R., L. Edman, et al. (2004). “Non equilibrium catalysis of single enzyme molecules.” Abstracts of Papers of the American Chemical Society 228: U201-U202 ;
Choi, B., G. Zocchi, et al. (2005). “Allosteric control through mechanical tension.” Physical Review Letters 95(7).

Principal investigator: David Bensimon (david[ at ]lps.ens.fr),
Address : ENS-LPS, 24 rue LHOMOND, Paris 75005, FRANCE
website: http://pimprenelle.lps.ens.fr/biolps/