Panasik Lab
Research
The Alpha/Beta Barrels
The eight stranded a/ b barrel protein fold is the largest family of protein structures (representing at least 10% of all known protein structures) and has the widest range of enzyme functions. My research focuses on elucidating the structural basis for folding specificity and thermodynamic stability in this class of enzymes and the future application of those principles in protein design.
We are currently investigating "Fold Specific" protein structure
adaptation strategies which suggest the pathways that a protein takes to
increased stability are specific to the specific structural
architecture of its polypeptide chain.
Directed Evolution Approach We use random PCR mutagenesis to create a library of genetic variants for a variety of enzymes including beta-galactosidase. These variants are placed in an expression host that is a beta-galactosidase negative yet positive for the lactose transport genes. A temperature selection is applied. Using chromogenic substrates for screening, or nutrient restriction for selection, variants that display the selected phenotype (activity or stability at higher or lower temperature) can be selected. These variants are subjected to future rounds of mutagenesis and selection until variants with dramatically different enzymatic properties are produced. Using multiple successes from these “Directed Evolution” experiments, coupled with careful analysis of structure, patterns of mutations begin to emerge. These patterns help us posit generalized, fold-specific, molecular mechanisms of protein structural adaptation to temperature. These principles are then tested using site directed mutagenesis and rational design.
Bioinformatics Approaches The Panasik lab
also looks at the same problem from a different angle through the lens
of Bioinformatics. Here, all extant psychrophilic, mesophilic, and
thermophilic alpha/beta barrel enzymes that have high resolution
crystal structures are annotated and compared. We analyze the
distribution of the structural features most commonly purported as the
molecular basis of thermostability; ion pairs, hydrogen bonds, solvent
accessible hydrophobic surface area buried upon folding, and
flexibility.
We find certain ion pairs interactions are
enriched in thermophilic alpha/beta barrel proteins. We will compare
these patterns observed in the Protein Data Bank (PDB) of all known 3D
protein structures to those patterns of adaptation that we see in our
directed evolution experiments.
An example of an ion pair interaction that is
more commonly found in thermophilic alpha/beta barrels.
Other Folds The above methods are applied to enzymes of other protein fold families to determine structural adaptation strategies to temperature that are specific to those folds.
Entropic Stabilization in
Thermostable Enzymes
Computational Biophysical Approaches. Using ab initio Monte Carlo simulations
programmed in PYTHON, the Panasik Lab estimates conformational space available to the unfolded state
of a peptide sequence and the conformational space that is disallowed due to steric exclusion of side chain and main chain atoms.
We propose novel classes of amino acid substitutions that increase
thermostability and have posited a new model of protein thermostability
through entropic stabilization.
where more non-native-like conformations
of the unfolded state are preferentially disallowed due to
atom-atom repulsions and steric clash in a thermostable sequence (shown
in red) as compared to a homologous mesophilic sequence (shown in blue).
More of the conformations ruled out due to steric clash for the
thermophilic sequence have higher RMSDs as compared to the native like
conformation for that sequence than mesophilic homologous sequences do.
Representations of Conformational Space:
The size of conformational space available to
the folded and unfolded states of a protein is not the only informative
feature in protein folding and stability. The type of
conformational space, i.e. which conformations are available and
not available are perhaps far more informative. In order to
differentiate between different local conformations of the protein
backbone it becomes nescessary to fnd a way to represent the entire
range of conformations in a manner that is easy to visualize and to
quantitate.
Above is a new 3D representation for multiple
conformations of tetrapeptide sequences called 'Dihedral Space'. Each
point represents a different tetrapeptide conformation. This 3D
representation of tetrapeptide conformations is similar to the 2D
Ramachandran plot for dipeptides. This figure is a sample of 10% of all
tetrapeptide conformations in the PDB.
Tetra peptide conformations found in a 60 x 60 x
60 degree cube of dihedral space called a "Hexostate" indicates that
similar regions in dihedral space correspond to similar conformations.
Graphic courtesy of Prof. N. Panasik, Jr., Claflin University.
Conformations that result when phi, psi angles
from dissallowed regions of the Ramachandran Plot cluster and inhabit
regions of dihedral space that are sparcely populated in the PDB.
Graphic courtesy of Prof. N. Panasik, Jr., Claflin University.
Dihedral Space can be divided into 60x60x60
cubes representing different conformations and the distribution of
conformations present in all proteins in the PDB can be analyzed.
Graphic courtesy of Prof. N. Panasik, Jr., Claflin University.
