Evolutionary Negative Design?

Ard LouisJonathan Doye, Michele Vendruscolo,

Janet Thornton

Why are proteins hard to crystallize?

Hypothesis: Proteins have evolved to avoid native state aggregation in vivo This “evolutionary negative design” also affects crystallization

experiments in vitro

Negative Design: The design of an object not to do something

Similar negative design principles in nature: Protein folding Non-native state aggregation (amyloid formation); different

evolutionary pressures Biological self-assembly (positive and negative design)

Negative design of protein folding against alternate configurations

C.M. Dobson, Nature 426, 884 (2003)

Levinthal Paradox: resolution relies on negative design in pathway.

Aside: Sequence space:20^100 = more atoms than exist in the universe

Negative design in mis-folded proteins; amyloid formation

C.M. Dobson, Nature 426, 884 (2003)

Copyright ©2002 by the National Academy of Sciences

Richardson, Jane S. and Richardson, David C. (2002) Proc. Natl. Acad. Sci. USA 99, 2754-2759

Fig. 3. (a-d) A set of equivalent, paired sheetedges from four different beta-sandwich protein families having thelectin/glucanase fold

Negative design in mis-folded proteins; amyloid formation

Copyright ©2002 by the National Academy of Sciences

Wang, Weixun and Hecht, Michael H. (2002) Proc. Natl. Acad. Sci. USA 99, 2760-2765

Fig. 1. (A) Schematic representation of a fibril formed byopen ended oligomerization of a six-stranded beta-sheet protein.beta-strands are shown in green, and turns in silver

Negative design in mis-folded proteins; amyloid formation

Normally self-assembly of surfactants etc… is very polydisperse

•Viruses•Nanotechnology?•Colloidal “molecules”?

Pauline Wong: to make these monodisperse icosohedra, the dimers must be designed out.

Positive and Negative Design for Self-Assembly

Protein crystallization is fundamentally different!(notwithstanding “crystallization slot”)

Colloids are artificially stablized (negative design against aggregation)

2. steric stabilization1. charge stabilization 3. nanohalo stabilization

Dynamic Nanoparticle Halos, S. Karanikas and AAL, Phys. Rev. Lett 93, 248303 (2004)

P. Pusey and W. van Megen, Nature 320,340 (1986)

Analogies with colloidal crystallization?

Cells are crowded environments

David Goodsell http://www.scripps.edu/mb/goodsell/gallery/patterson.html

proteins in blue, DNA and RNA in red and orange, lipids in yellow, and carbohydrates green Ribosomes are colored magenta.

Cells are crowded environments

Network of protein-protein interactions must have bothSpecific functional interactions (> 100) (positive design)Generic repulsion to avoid aggregation (negative design)

Remarkable control of this interactome -- many constraints!

With colloids such high concentrations would result in gunk,

….unless you stabilize them

Negative design and protein crystallization

Most likely through surface properties•Surface residues are not random (J. Thornton)

Key question: are the design mechanisms against aggregation robust enough to affect protein crystallization in vitro?(are you fighting evolution?)

Yes: Evidence? •Protein as a variable (G.E. Dale, C. Oefner & A. D’Arcy, J. Struc. Biol 142, 88 (2003))•Random mutagenesis of surface residues•Directed mutagenesis of Lys etc… (Derewenda)•In-vivo crystallization; when nature wants to it can

How do proteins achieve negative design?

• Other study: Human thymidylate synthase:11 surface mutations: most led to enhanced crystallizability (McElroy et al J. Crys. Growth 122, 265 (1992))

DNA gyrase B subunit from E-coli. Crystals grown in the presence of novobiocin; (a)-(c) crystals from original microbatch,(d) optimized for X-ray analysis.(a) Wild type,(b) K14R/F104Y mutant, (c) K57R/I82N mutant,(d) F104Y mutant.

Mutagenesis studies (protein as a variable)

A. D'Arcy, M. Stihle, D.Kostrewa and G. Dale,Acta Cryst. D 55, 1623 (1999)

Rational mutagenesis studies

Derewenda group: Mutations of Lys -> Ala etc….

Is Lysine like a steric stabilizer (entropy)?

Role of water??

Mechanism?

Protein crystallization in vivo

Protein crystallization, and more generally native state aggregation, is likely to be harmful to the cell

Examples•Cataracts: crystallization and aggregation of the gamma crystallins•Sickle-cell anaemia: ordered aggregation of hemoglobin•Hemoglobin C disease: crystallization of hemoglobin•(many more in A. McPherson’s book)

Non-native state aggregation is more common in part because it is harder for evolution to control disordered proteins. Also, many of these diseases may be post-evolutionary (C. Dobson) and related to the breakdown of regulatory machinery.

J. Doye

•Protein crystallization in vivo

Storage•Seed proteins•Insulin granules•Baccillus thuringiensis•Ribosome crystals in hibernation animals•Crystals in egg yolks

Encapsulation•Baculoviruses

Obstruction•Hex-1 protein crystals in Woronin bodies in fungi•Serum albumim crystals at wound?

Accidental?•Iridoviruses•Inclusion bodies in expression systems

•Baculoviruses etc.. Virus-insect relationships

Encapsulated virus rod of P. interpunctella (army worm) granulovirus

Virus-insect relationshipsKenneth M. SmithLongman (New York 1976)

“this book has been written by one of the few remaining old-fashioned virologists…”

•Baculoviruses etc.. Virus-insect relationships

Nuclear Polyhedron (NPV)(micron size crystals)

Cytoplastic Polyhedron (CPV)

Granulovirus (Smaller crystals)

Release of a viron in alkali

“Wipfelkrankheit” in catepillars“Tree-top disease”“catepillar wilt”

•Baculoviruses etc.. Virus-insect relationships

Insect cells full of baculovirus containing polyhedra

“Wipfelkrankheit” in catepillars“Tree-top disease”“catepillar wilt”

Nuclear Polyhedron (NPV)(micron size crystals)

Cytoplastic Polyhedron (CPV)

Granulovirus (Smaller crystals)

•Baculoviruses etc.. Virus-insect relationships

Relatively monodisperse polyhedral crystals associated with Anopholes quadrimaculatus CPV

•Bacillus Thuringiensis

Insectial bacterium (commonly known as Bt)Production of protein toxins are associated with sporulation.Crystals involve a significant number of interprotein disulphide bridgesCrystals dissolve in alkaline environment of the insect gut, and aid entry

of germinating spores into the host

•Serum Albumin

“some wounds in the blood plasma harden in air to form crystals over the wound. Other blood proteins then help form a clot over the wound, preventing excessive blood loss.

•Serum Albumin

Proteins bodies in seeds

Square inprint of protein crystal evident in freeze-fracture of squash cotyledon

•Peroxisomes

Crystals of oxidative ensymes (catalase and urate oxidase) from a Peroxisome. Suggestion that these are storage crystals to guarantee a stable minimum level of catalase activity in the antioxidative system of plant cells.

•Conclusions

•Negative Design is ubiquitous in nature

•Understanding the mechanisms of this negative design may lead to rational methods for protein crystallization that seek to overcome this

•You’re probably fighting evolution

•Protein crystallization in vivo shows that low crystallizability is not an intrinsic property of proteins

•Future directions

with Janet Thornton, Jonathan Doye and Michele VendruscoloEBI and Dept. of Chemistry, Cambridge U.

J.P.K. Doye, A.A. Louis and M. Vendruscolo,Inhibition of protein crystallization by evolutionary negative design, Phys. Bio 1, P9-p113 (2004)

Combination of bioinformatics plus modelling approaches at various length scales

•Crystal contacts <--> crystal structures • role of mutations•Frustration•Minimal models

•Lysine etc …. (why so disfavoured at contacts?)•H20 and polar/hydrophobic surface patterns

We greatly value your input!

Also, we are collecting examples of in-vivo crystallisation

2-D simple models of randomness