Structure/function relationship in proteins Principal investigator: David Bensimon (david@lps.ens.fr), Address : ENS-LPS, 24 rue LHOMOND, Paris 75005, 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). 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).

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

Numerical simulations on biological macromolecules are carried out in the Laboratoire de Biochimie Théorique (denoted LBT). This group has more than ten years of experience in developing and applying modeling methods to study the deformation of biological macromolecules [19-23]. Besides a strong publication activity, LBT participates in National and European projects like the ANR project FonFlon and the EU project IMMUNOPRION. LBT has considerable experience in advanced simulation methods based on molecular dynamics simulations of challenging biological systems like membrane proteins with up to 340 000 atoms. As such systems require long simulation timescales, LBT is an important user of the French national IDRIS computing center and familiar with high performance computing and parallelized codes on PC clusters or supercomputers. Furthermore simplified protein models (going from a full atomic description to one or two points per residue only) are being developed and evaluated in order to use modern multi-scale approaches for efficient simulation of complex systems, even on commodity hardware.

Current research topics like the investigation of mechanical properties of proteins or macromolecular docking are particularly apt to virtual reality extensions. An interactive graphics system with a haptic interface, an early FVNano prototype named SHAMAN, was already developed and aims at interfacing the numerous in-house software packages [58,59]. In particular, LBT has developed software based on internal coordinates reducing the number of variables taken into account during modeling and allowing the use of energy minimization for the study of large conformational changes [20, 21].

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R20. Lafontaine, I. and R. Lavery, Collective Variable Modelling of Nucleic Acids. Curr. Opin. Struct. Biol., 1999. 76: p. 2760-2768.

R21. Lavery, R., Modeling Nucleic Acids: Fine Structure, Flexibility and Conformational Transitions. Adv. Comput. Biol., 1994. 1: p. 69-145.

R22. Lavery, R., et al., Structure and mechanics of single biomolecules: experiment and simulation. J. Phys.: Condens. Matter, 2002. 14: p. R383-R414.

R23. Lebrun, A., R. Lavery, and H. Weinstein, Modeling multi-component protein-DNA complexes: the role of bending and dimerization in the complex of p53 dimers with DNA. Protein Engineering, 2001. 14: p. 233-243.

R58. M. Baaden, E. Gasser, C. Prévost, R. Lavery, SHAMAN – Système HAptique de MAnipulation Nanoscopique. http://www.ibpc.fr/UPR9080/axes_recherches1.html

R59. E. Gasser, SHAMAN – Manipulation sensitive des macromolécules, Rapport DESS, Aout 2003