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