Download Example 2 - The Graduate School | UNC Charlotte

FAST Predictions for Protein Flexibility and Stability
Dennis R. Livesay and Donald J. Jacobs
The University of North Carolina at Charlotte
Linking Protein Flexibility to Thermodynamic Stability
Computer-aided Protein Engineering and Design
MECHANICS
Globally Rigid
Globally Flexible
What is the motivation?
Computational design of proteins with specific stability and flexibility relationships
would significantly advance the pharmaceutical and biotechnology industries. Yet,
many pitfalls continue to limit the success of computer-aided molecular design.
-TS
Why focus on conformational flexibility?
A critical link between structure, stability and biological function is conformational
flexibility, which is characterized by an ensemble of accessible states.
Conformational flexibility regulates specificity in mechanical response as manifested
in dynamics. For example, enzymes must be flexible enough to mediate a reaction
pathway, yet rigid enough to achieve molecular recognition.
Conformational flexibility regulates protein stability through conformational entropy
as an enthalpy-entropy compensation mechanism. For example, the binding affinity
of a ligand at a receptor site in a protein can be altered by the binding of a different
molecule at a distant site, leading to allostery.
H
H
-TS
-TS
H
STABLE representing the
native state (NS)
UNSTABLE representing the
transition state (TS)
STABLE representing the
unfolded state (US)
THERMODYNAMICS
QSFR: Quantitative Stability & Flexibility Relationships
What is the challenge?
The challenge is to develop a physical model to accurately predict protein flexibility
and stability for specified thermodynamic and solvent conditions in fast computing
times for high throughput applications.
Simulation, Models and Fundamental Science
Assuming a perfect model, simulations will generally miss important mechanisms
because of incomplete exploration of the ensemble of accessible states. To overcome
this problem it is common to employ the simplest possible model that retains all
essential elements of interest. However, simulations based on a flawed model will
predict events that are impossible to occur. That is, an oversimplified model will
misrepresent the underlying Physics, Chemistry and/or Biology.
(a) Free energy landscapes for E. coli & T. thermophilius orthologs at their respective
Tm. (b) Rigid cluster size susceptibility. (c) Probability metric for backbone flexibility.
Comparative QSFR Across Protein Families
A Mechanical Model for Protein Thermodynamics
A protein consist of many types of competing weak interactions that determine its
structure and thermodynamic stability. The most fundamental aspect of any model is
to identify all relevant degrees of freedom and constraints. In statistical physics,
global constraints such as fixed total energy or temperature, or fixed total volume or
pressure, play an essential role in defining the relevant thermodynamic ensemble to
calculate equilibrium properties. Likewise, chemical bonding and weaker contact
interactions act as mechanical constraints. Applying constraint theory allows the
ensemble of accessible states to be partitioned in terms of mechanical characteristics.
A Radically Different Computational Method
(Top) Sequence alignment of 9 oxidized thioredoxin
(TRX) structures. (Red, blue) indicates the backbone is
(flexible, rigid). (Right) A dendrogram describing the
clustering of 9 TRX cooperativity correlation.
Mapping out allosteric response in Calmodulin (CaM)
What is the big picture?
A free energy functional is derived by applying constraint theory to a free energy
decomposition scheme. Each interaction type is modeled by a molecular partition
function (MPF). Free energy reconstitution is the process of solving the functional
using graph rigidity to account for nonadditivity in conformational entropy. Bare
parameters in a MPF describe an interaction within a reference environment. The
MPF parameters renormalize to reflect a modified environment that is determined
heat
self-consistently. Consequently, the actual enthalpy/entropy contributions depend
resistor
on many-body effects and the thermodynamic/solvent conditions. Order parameters
describing solvation and conformational flexibility define a free energy landscape
from which thermodynamic properties are calculated. Mechanical properties are
obtained from graph rigidity calculations that are appropriately ensemble averaged.
What is the significance?
Thermodynamic and mechanical properties are calculated in matter of minutes using
FAST software that provides a Flexibility And Stability Test on aqueous proteins.
Quantitative Stability and Flexibility Relationships (QSFR) are calculated with
great precision and can be employed in high throughput applications.
(Top) Response in conformational flexibility to
a mechanical perturbation at Ile136 (shown in
orange). (Red, blue) indicates a (degrease,
increase) in flexibility. (Right) The complete
perturbation/response characteristics.
FAST is now in Development: Expected Release is 2010
The extended DCM includes solvation effects, and will be able to accurately decompose the changes
in free energy due to different mechanisms. In the new model, pressure, pH and solute concentrations
are accounted for, and mobility information will be added to the QSFR descriptions.
This work is supported by: NIH R01 GM070382
Contact information: [email protected] or [email protected]