![Page 1: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/1.jpg)
Design of a novel globular protein with atomic-level
accuracy
![Page 2: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/2.jpg)
NOVEL METHODS
• Computational methods
• New developments in the making
• Opening of a new field of science
![Page 3: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/3.jpg)
Ideas
• Redesign naturally occurring proteins so that they have enhanced stability or new functionality
![Page 4: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/4.jpg)
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 protein with topology that is not present in the Protein Structure Data Base
![Page 5: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/5.jpg)
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
![Page 6: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/6.jpg)
Some more Top7 features
• Highly soluble protein
• Monomeric structure
• Thermally stable
• More stable than most proteins of its size
![Page 7: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/7.jpg)
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
![Page 8: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/8.jpg)
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
![Page 9: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/9.jpg)
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
![Page 10: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/10.jpg)
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
![Page 11: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/11.jpg)
Myoglobin/Hemoglobin
![Page 12: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/12.jpg)
Globin Fold
• Made up of alpha helices
• Hemoglobin and myoglobin are examples
![Page 13: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/13.jpg)
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
![Page 14: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/14.jpg)
Hemoglobin
• Oxygen binds to heme group
• No oxygen bound called T state
• Oxygen bound called R
![Page 15: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/15.jpg)
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 firstThe others will bind much easier
![Page 16: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/16.jpg)
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
![Page 17: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/17.jpg)
Enzymes
• Enzymes speeds things up substantially
![Page 18: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/18.jpg)
Enzymes
• Have high specificity for their substrate
![Page 19: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/19.jpg)
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
![Page 20: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/20.jpg)
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
![Page 21: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/21.jpg)
Serine Proteases
![Page 22: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/22.jpg)
4 aspects for serine proteases
• Catalytic Triad
• Oxyanion hole
• Specific binding
• Non-specific binding
![Page 23: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/23.jpg)
Catalytic TriadAlways 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
![Page 24: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/24.jpg)
Continued
His residue accepts proton from reactive serine and helps stabalize the transition state
Ser forms covalent bond with substrate
![Page 25: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/25.jpg)
Oxyanion holeVery 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
![Page 26: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/26.jpg)
Specificity binding pocket
Accepts different side chains from different substrates dependingOn the member of the serine protease family
![Page 27: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/27.jpg)
Specificity Pocket
![Page 28: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/28.jpg)
Specificity PocketChymotrypsin cleaves bulky aromatic side chains: Serine at the Bottom of the pocket which wont interfere too much with largeAromatic side chain
Trypsin cleaves next to large positively charged side chains; AspAt bottom which will attract positive side chain from substrate
Elastase cleaves small uncharged sidechains:
![Page 29: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/29.jpg)
Two Phase Reaction
First: acylation in which the peptide bond gets cleaved
When the substrate comes close, the oxygen of serine 195Bends to interact with substrateRESULTS in bond between the reactive serineAnd 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
![Page 30: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/30.jpg)
First Phase
![Page 31: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/31.jpg)
Continued
This then allows for peptide to be released and the end of the first step
![Page 32: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/32.jpg)
Phase 2
Enzyme still attached to remaining part of peptide
Deacylation: water comes along and forms a new tetrahedralTransition state which is also unstable
A covalent bond is formed between C and O and the fragment isreleased
Hydrgoen then goes back to serine and enzyme can be used again
![Page 33: Design of a novel globular protein with atomic-level accuracy](https://reader036.vdocument.in/reader036/viewer/2022062516/56649d815503460f94a65e5b/html5/thumbnails/33.jpg)
Second phase