Transcript Design of a novel globularprotein with atommic

Design of a novel globular
protein with atomic-level
accuracy
NOVEL METHODS
• Computational methods
• New developments in the making
• Opening of a new field of science
Ideas
• Redesign naturally occurring proteins so
that they have enhanced stability or new
functionality
This group
• Found a procedure for the development of
low free energy structures
• Led them to the creation of the protein
TOP7
• A 93 residue a/b protein with topology that
is not present in the Protein Structure Data
Base
Design Protocol
• Critical is cycling between seequence
design and backbone optimization. The goal
is to find the lowest free energy backbone
conformation for a fixed amino acid
sequence
Some more Top7 features
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Highly soluble protein
Monomeric structure
Thermally stable
More stable than most proteins of its size
Conclusion
• Top7 shows that the design of globular
proteins not yet observed in nature is
possible but can be extremely stable
• The methods used to design TOp7 are
applicable to any globular protein structure
• This may open the door for exploration of
new protein strucutres and architectures
A few more signaling things
• EGF receptor dimerizes when ligand binds
and then causes signaling cascade for cell
division
uses covalent linkage of phosphate to
downstream proteins to carry on signal
Continued
• SH molecules (SH2 SH3) are adaptor
proteins.
• They will recognize specific parts of target
molecule and bind and now the phosphate
will be transferred on to the target molecule
Calmodulin
• Helix loop helix (EF Hand)
• Important in many signal transduction
pathways
• Charged glu and asp bind Ca2+
• Unbound state resembles a dumbbell
• Bound state it becomes very compact
Myoglobin/Hemoglobin
Globin Fold
• Made up of alpha helices
• Hemoglobin and myoglobin are examples
Oxygen is not soluble in blood so
it needs carriers
• Myoglobin
• One subunit
• Transports oxygen in
muscle
• Hemoglobin
• 4 subunits
• Transports oxygen all
over body
• Four oxygen binding
sites
Hemoglobin
• Oxygen binds to heme group
• No oxygen bound called T state
• Oxygen bound called R
Binding of Oxygen
• When shifts from T to R the iron moves to
the center of the heme plane because an
overall structural shift. This shift then gets
propagated throughout the structure and
leads to the change in other subunits
COOPERATIVE BINDING: with the binding of the first
The others will bind much easier
Sickle Cell
• Base pair change in hemoglobin from glu to
val
Makes hemoglobin sticky and red blood cells
will clump together and cause all sorts of
problems
Enzymes
• Enzymes speeds things up substantially
Enzymes
• Have high specificity for their substrate
How they speed things up
• Increase substrate at catalytic site
• Physically bring molecules to the right
place and increase local concentration
• Selectively stabalize by binding to
TRANSITION STATE
• Lower the activation energy by stabalizing
the transition state
ACTIVE SITE
• Pocket or groove lined with specific amino
acid residues
• Here is where the substrate goes and
undergoes some sort of chemical
transformation
• Therefore it can be seen that the enzyme is
playing the role in this chemical
modification
Serine Proteases
4 aspects for serine proteases
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Catalytic Triad
Oxyanion hole
Specific binding
Non-specific binding
Catalytic Triad
Always see these three particular side chains:
ASP 102, His 57, and Ser 195
All spread out in primary sequence
Come together at active site in binding pocket
Continued
His residue accepts proton from reactive serine
and helps stabalize the transition state
Ser forms covalent bond with substrate
Oxyanion hole
Very important in stabalizing the transition state by
Forming H Bonds
Allows for nonspecific substrate binding to the main
chain of the substrate
This is the region of the enzyme that is non specific
Specific parts are taken care of with the specificity
pocket
Specificity binding pocket
Accepts different side chains from different substrates depending
On the member of the serine protease family
Specificity Pocket
Specificity Pocket
Chymotrypsin cleaves bulky aromatic side chains: Serine at the
Bottom of the pocket which wont interfere too much with large
Aromatic side chain
Trypsin cleaves next to large positively charged side chains; Asp
At bottom which will attract positive side chain from substrate
Elastase cleaves small uncharged sidechains:
Two Phase Reaction
First: acylation in which the peptide bond gets cleaved
When the substrate comes close, the oxygen of serine 195
Bends to interact with substrate
RESULTS in bond between the reactive serine
And carboxly or substrate
Proton of serine is donated to His
Peptide bond goes from planar to tetrahedral state
This state is unstable and very short lived
First Phase
Continued
This then allows for peptide to be released
and the end of the first step
Phase 2
Enzyme still attached to remaining part of peptide
Deacylation: water comes along and forms a new tetrahedral
Transition state which is also unstable
A covalent bond is formed between C and O and the fragment is
released
Hydrgoen then goes back to serine and enzyme can be used again
Second phase