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Second Genetic Code:
Sequence StructureFolding
Design(reverse folding)
Computer Algorithm
Input Output
Folding Sequence Structure
Design Structure Sequence
Protein Stability (thermal)• Protein engineering (mutagenesis)1. S-S bridges
a. -CH2-S-S-CH2-b. Analysis of all possibilities (many)c. Energy minimization to reduce to a few plausible candidatesd. Site-selective mutationse. Protein synthesisf. Assay:
example – T4 lysozyme (x-ray structure known)Reducing degrees of freedom (entropy) increases protein stability
Protein Stability Cont…
2. Gly and Pro-Gly freedom-Pro Constraints (side chain is fixed by covalent bond to main chain- Gly Pro has propensity to increase stability (more delicate)- GlyAla usually increase- ProAla usually decrease
Protein Stability Cont…
3. Dipolar stabilityN-end (-a.a.)
C-end (+a.a.)increase stability by mutating residues at N-
end of helices from polar to negative (e.g. ASNASP, SERASP)
Helix:
Protein Stability4. Hydrophobicity in the core (cavity)
-Barnase (bacterial RNAse-110 a.a.)-structure by both x-ray and NMR
-introducing cavities in the core by mutations such as IleVal or PheLeu
Cavity for a CH2
Stability by 1kcal/mol
-More delicate design-Needs structure
Agonist of the erythropoietin receptor identified from peptide libraries
Prediction of Structure From Sequence
• Empirical – in progress• ~75% successful-at best (62-65%). For the membrane-
embedded domains of membrane proteins up to 90%• Essence: Pattern Recognition• Key: Evolutionary Information
– Sequence homology implies similarity in structure and function– By inference/By Anaysis
• Data bases (2007 >500,000 seq., 2015 >107,000 Structures
• Information: Prediction• Example: Homologous proteins
Conserved Core Variable Loop
Secondary Structure Prediction for 3-Model
• Predict: α, β, loop, β-turn• Predict: membrane-spanning α-helix• Predict: Amphipatic structures
α β• Prediction of the folded structure of
tryptophan synthetase, and• the catalytic subunit of c-AMP dependent
protein kinase
Chou & Fassman (1974)• Frequency of occurrence of a given a.a. in α, β,
and loops in all protein structures in the database (statistical)
• Nearest neighbors• output: probability for each residue to be in α,
β, or Loop• Artificial intelligence/neural networks
– Train a computer to recognize patterns – the more information and the “more practice” the higher the accuracy (in progress)
Bacterial Photosynthetic Reaction Center
Bacterial Photosynthetic Reaction Center
Protein Codesaa Sequence, 1D
Structure, 3D
REVERSE FOLDING(design)
FOLDING
http://www.npaci.edu/enVision/v15.4/images/proteinfolding1.jp
Design
• Minibody• Chymohelizyme• Calcium sensor• Acetylcholine Receptor Channel
Minibody
• 61 residue synthetic peptide• All • Template: Heavy chain variable domain of
the immunoglobulin• Hypervariable loops• Binding site: Histidines in each
hypervariable loop• The protein folds and binds Zn2+
• Nature 362: March 25, 1993
Chymohelizyme• Design: Computer-assisted protein design• Four helix bundle – parallel, amphipathic• Serine protease catalytic triad –Ser, His, Asp at
the N-end of the four-helix bundle in the same spatial arrangement as chymotrypsin
• Oxyanion hole and substrate binding pocket for acetyltyrosineethylester, a classical substrate of CT were included in the design
• Synthetic enzyme folds, is catalytically active and sensitive to a specific inhibitor
• Science 248:1544, 1990)
Channel Design
REVERSE FOLDINGDesign
Sequence?
The Acetylcholine Receptor• Nicotinic acetylcholine receptor: A pentamer
– Ion channel for influx of Na+, Ca2+– Gate opened by acetylcholine
Channel Design
REVERSE FOLDING
EKMSTAISVLLAQAVFLLLTSQR ?
design
The Acetylcholine Receptor: Pentamer
M2 HELICES
Channel Design
REVERSE FOLDING
EKMSTAISVLLAQAVFLLLTSQR ?
design
M2 Channels:Pentamer (T5M2)
K*AK*KK*PEK*EK*G
* = M2 = EKMSTAISVLLAQAVFLLLTSQR
Montal et al. Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor. FEBS Lett. 1993, 209(3): p. 261-266.
NMR:Structure and Orientation
Opella et al. Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy. Nat. Struct. Biol., 1999; 6(4): p. 374-9.
NMR STRUCTURE MODEL
Opella et al. Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy. Nat. Struct. Biol., 1999; 6(4): p. 374-9.
Moving closer?
Unwin, N. Refined Structure of the Nicotinic Acetylcholine Receptor at 4A Resolution. J. Mol. Biol., 2005; 346(4): p. 967-89.
2003 2005
Acetylcholine Receptor Structure @ 4 Å in 2005
Unwin, N. Refined Structure of the Nicotinic Acetylcholine Receptor at 4A Resolution. J. Mol. Biol., 2005; 346(4): p. 967-89.
Channel Design
REVERSE FOLDING
EKMSTAISVLLAQAVFLLLTSQR
FOLDING design
Channel Design
REVERSE FOLDING
EKMSTAISVLLAQAVFLLLTSQR ?
design
Protein Codesaa Sequence, 1D
Structure, 3D
REVERSE FOLDING(design)
FOLDING
http://www.npaci.edu/enVision/v15.4/images/proteinfolding1.jp