Chapter 17. Prediction, Engineering, and Design of Protein Structures

Transcription

1 Chapter 17. Prediction, Engineering, and Design of Protein Structures

2 Homologous proteins have similar structure and function Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.349.

3 Homologous proteins have conserved structural cores and variable loop regions Song and Suh (1998) J. Mol. Biol., 275,

4 Prediction vs Experiment (CDR regions of a mab) Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.350.

5 Knowledge of secondary structure in necessary for prediction of tertiary structure Prediction methods for secondary structure benefit from multiple alignment of homologous proteins Many different amino acid sequences give similar three-dimensional structures Prediction of protein structure from sequence is an unsolved problem Threading methods can assign amino acid sequences to known three-dimensional folds

6 Proteins can be made more stable by engineering Model protein: T4 lysozyme (1) Reducing the difference in entropy between folded and unfolded protein, which in practice means reducing the number of conformations in folded state (2) Stabilizing the α-helices (3) Increasing the number of hydrophobic interactions in the interior core

7 Thermostable α-amylase from B. licheniformis Temperature resistance profiles of wildtype BLA (*, dotted line) and its variants: 264S-265Y (Δ); 172R ( ); 133I-209V ( );133I-190F-209V ( );133I-190F- 209V-264S-265Y ( );133I-156Y-172R- 181T-190F-209V-264S-265Y ( ); H133I- H156Y-A181T-N190F-A209V-Q264S- N265Y ( ); 237D (x); 204K (+); 204K- D237D ({star}v) Declerck, N. et al. Protein Eng :

8 Disulfide bridges increases protein stability Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.355.

9 Disulfide bridges increases protein stability Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.356.

10 Disulfide bridges increases protein stability Tm value (4.8 C C C ~ 22 C) Glycine and proline have opposite effects on stability << Entropy terms >>

11 Stabilizing the dipoles of α-helices increase stability Each mutant: Tm ~ 2 C Mutants that fill cavities in hydrophobic cores do not stabilize T4 lysozyme Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.357.

12 Proteins can be engineered by combinational methods Library Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.359.

13 Phage display links the protein library to DNA Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.360.

14 Affinity and specificity of proteinase inhibitors can be optimized by phage display Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.361.

15 Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.362.

16 Structural scaffolds can be reduced in size while function in retained Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.363.

17 Phage display of random peptide libraries identified agonists of erythropoietin receptor Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.365.

18 DNA shuffling allows accelerated evolution of genes Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.366.

19 Directed evolution is a method used in protein engineering to harn ess the power of Darwinian selection to evolve proteins or RNA wi th desirable properties not found in nature. A typical directed evolution experiment involves three steps: Diversification: The gene encoding the protein of interest is mutated and/or reco mbined at random to create a large library of gene variants. Techniques com monly used in this step are error-prone PCR and DNA shuffling. Selection: The library is tested for the presence of mutants (variants) possessing the desired property using a screen or selection. Screens enable the researche r to identify and isolate high-performing mutants by hand, while selections au tomatically eliminate all nonfunctional mutants. Amplification: The variants identified in the selection or screen are replicated m any-fold, enabling researchers to sequence their DNA in order to understand what mutations have occurred.

20 Protein structures can be designed from first principles Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.367.

21 Protein structures can be designed from first principles Is it possible to design from first principles a sequence whose main chain obtains this zinc finger fold without zinc atom to stabilize the structure? Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.176, 178.

22 Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.368.

23 A β structure has been converted to an α structure by changing only half of the sequence Paracelsus challenge (a prize of $1000) in 1994 Converting one protein fold into another while retaining 50 % of the original sequence Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.369.

24 Janus vs B1 (from Protein G) 50% identity Similarity to Rop % 41% 28/56 Branden & Tooze (1998), Introduction to protein structure, 2 nd ed., p.369.