Postdoc Offer Structure Function Relationships In Proteins Using Single Molecule Fluorescence

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).