de novo Protein Design

download report

Transcript de novo Protein Design

de novo Protein Design
Presented by
Alison Fraser, Christine Lee,
Pradhuman Jhala, Corban Rivera
Outline




Introduction
Computational methods used for sequence
and structure
Biophysical and structural characteristics
of novel protein
Conclusion
Introduction




Number of protein folds
Computational methods for identifying
amino acid sequences compatible with
target structure – not for protein creation
Side Chains as Templates
New protein design => more rigorous test
of current force fields and optimization
methodology than redesign of naturally
occurring proteins
Introduction (continued)




Search of nearby conformational space
and sequence space
2 methods of protein redesign (variation
of backbone conformation and amino acid
sequence)
Development of procedure for identifying
low free energy sequence-structure pairs
that iterates between sequence
optimization and structure prediction
Result: 93 residue protein with topology
not in PDB
Structure to Sequence


RosettaDesign predicts a amino acid
sequence from a desired structure.
Input and Output


Input a structure you would like to create
Output a amino acid sequence that will
produce the structure with low free energy
Sequence to Structure


RosettaDesign is used to predict protein
structure from protein residue sequence.
Input and Output


Input Amino Acid sequence
Output a Predicted near minimum free energy
structure
How does Top7 compare to
proteins in nature?



Folding
Stability
Structure
Stability

Thermally stable

CD Spectrum at 98˚C
is nearly
indistinguishable from
that at 25˚C
Folding of Top7


At intermediate
concentrations (~5 M) of
guanidine hydrochloride
(GuHCl) Top7 unfolds
cooperatively
Steep transition in
chemical denaturation is
characteristic of the twostate unfolding expected
for small, two-state,
monomeric single-domain
protein
Structure

Nuclear Overhauser
effect spectroscopy
(NOESY) and
heteronuclear single
quantum coherence
(HSQC) exhibit
features characteristic
of protein with
substantial beta-sheet
content
Comparing Top7 to model

Crystallization


Top7 yielded crystals that diffracted to 2.5 Å
Strong molecular replacement (MR) solution
to phase problem


This suggest design model very close to true
structure
Top7 crystal like the model was also
judged to be a novel topology by TOPS
server
Comparison of model (blue) and
the solved x-ray structure (red)
Implications


Atomic Level Accuracy
(RMSD = 1.17 oA) in de
novo Protein Design
Validation of Accuracy of
Potential Functions
Reasons

Optimization of Sequence and Structure

No Functional Constraints

Extensive Optimization

No kinetics
Possible Future Impacts


Synthetic proteins
Protein Therapeutics and Molecular
Mechanics