Publications

Please email reprint requests to Dek Woolfson at dek@biols.susx.ac.uk.
47 . ZiCo: A Peptide Designed to Switch Folded State upon Binding Zinc.
E Cerasoli, BK Sharpe, DN Woolfson.
J. Am. Chem. Soc. 127 15008-9 (2005) Abstract)
46 . The design of coiled-coil structures and assemblies
DN Woolfson
Adv Protein Chem 70 79-112 (2005) Abstract)
45 . MaP peptides: Programming the self-assembly of peptide-based mesoscopic matrices
MG Ryadnov, DN Woolfson
J. Am. Chem. Soc 127 12407-12415 (2005) Abstract)
44 . Polar assembly in a designed protein fiber
AM Smith, SFA Acquah, N Bone, HW Kroto, MG Ryadnov, MSP Stevens, DRM Walton, & DN Woolfson*
Angew. Chem. Int. Ed. 44 325-328 (2005) Abstract)
43 . Sequence and Structural Duality: Designing Peptides to Adopt Two Stable Conformations
MJ Pandya, E Cerasoli, A Joseph, RG, E Waite & DN Woolfson*
J. Am. Chem. Soc 126 17016-17024 (2004) Abstract)
42 . Design and synthesis of a nitrogen-mustard derivative stabilised by apo-Neocarzinostatin
MD Urbaniak, JP Bingham, JA Hartley, DN Woolfson & S Caddick*
J Med Chem 47 4710-4715 (2004) Abstract)
41 . Biophysical and mutational analysis of the putative bZIP domain of Epstein-Barr virus EBNA 3C.
MJ West*, HM Webb, AJ Sinclair & DN Woolfson
J Virology 78 9431-9445 (2004) Abstract)
40 . Fiber Recruiting (FiRe) Peptides: Non-Covalent Decoration of an Engineered Protein Scaffold
MG Ryadnov and DN Woolfson*
J Am Chem Soc 126 7454-7455 (2004) Abstract)
39 . Engineered and Designed Peptide-based Fibrous Biomaterials CE MacPhee & DN Woolfson*
CE MacPhee & DN Woolfson*
Current Opinion in Solid State and Material Science 8 2 141-149 (2004) Abstract)
38 . Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin
CG Benitez-Cardoza, K Stott, M Hirshberg, DN Woolfson & SE Jackson*
Biochemistry 43 5195-5203 (2004) Abstract)
37 . Extended knobs-into-holes packing in classical and complex coiled-coil assemblies
John Walshaw and Derek N. Woolfson
Journal of Structural Biology 144 3 349-361 (2003) Abstract)
36 . "Belt and Braces": A Peptide-Based Linker System of de Novo Design
MG Ryadnov, B Ceyhan, CM Niemeyer & DN Woolfson
J Amer Chem Soc 125 31 9388-9394 (2003) Abstract)
35 . Introducing Branches into a Self-Assembling Peptide Fiber
MG Ryadnov & DN Woolfson
Angew. Chem. - Int. Ed. 42 3021-3023 (2003) Abstract)
34 . Engineering the morphology of a self-assembling protein fibre
MG Ryadnov & DN Woolfson
Nature Materials 2 5 329-332 (2003) (Abstract)
33 . Chemical Synthesis and Cytotoxicity of Dihydroxylated Cyclopentenone Analogues of Neocarzinostatin Chromophore
MD Urbaniak, LM Frost, JP Bingham, LR Kelland, JA Hartley, DN Woolfson & S Caddick
Bioorganic & Medicinal Chemistry Letters 13 12 2025 - 2027 (2003) (Abstract)
32. Solution Structure of a Novel Chromoprotein Derived from Apo-Neocarzinostatin and a Synthetic Chromophore.
Michael D. Urbaniak, Frederick W. Muskett, Michael D. Finucane, Stephen Caddick and Derek N. Woolfson
Biochemistry 41 11731-11739 (2002) (Abstract)
31 . Mini-proteins Trp the light fantastic
Gellman SH, Woolfson DN
Nat Struct Biol 9 6 408-410 (2002) (Abstract)
30 . Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37
Siligardi, G; Panaretou, B; Meyer, P; Singh, S; Woolfson, DN; Piper, PW; Pearl, LH; Prodromou, C.
J. Biol. Chem 277 23 20151-20159 (2002) (Abstract)
29 . Generalised Crick equations for modelling non-canonical coiled coils
Offer, G; Hicks, MR; Woolfson, DN;
J. Struct. Biol. 137 41-53 (2002) (Abstract)
28 . Investigating the tolerance of coiled-coil peptides to non-heptad sequence inserts
Hicks, MR; Walshaw, J; Woolfson, DN;
J. Struct. Biol. 137 73-81 (2002) (Abstract)
27 . A designed system for assessing how sequence affects alpha-to-beta conformational transitions in proteins
Ciani, B; Hutchinson, EG; Sessions, RB; Woolfson, DN.
J. Biol. Chem 277 10150-10155 (2002) (Abstract)
26 . Core-directed Protein Design
Woolfson, DN.
Curr. Opin. Struct. Biol. 11 464-471 (2001) (Abstract)
25 . Guidelines For the Assembly of Novel Coiled-coil Structures:alpha-sheets and Alpha-cylinders.
Walshaw, J; Shipway, JM; Woolfson, DN..
Biochem Soc Symposium 68, "From Protein Folding to New Enzymes" Eds. A.Berry & SE Radford 111-123 (2001) (Abstract)
24 . Biophysical Analysis of Natural Variants of the Multimerization Region of Epstein-barr Virus Lytic-switch Protein Bzlf1
Hicks, MR; Balesaria, S; Medina-Palazon, C;Pandya, MJ; Woolfson, DN; Sinclair, AJ.
Journal of Virology 75 5381-5384 (2001) (Abstract)
23 . Socket: a Program For Identifying and Analysing Coiled-coil Motifs Within Protein Structures
Walshaw, J; Woolfson, DN.
Journal of Molecular Biology 307 1427-1450 (2001) (Abstract)
22 . Open-and-shut Cases in Coiled-coil Assembly: Alpha-sheets and Alpha-cylinders
Walshaw, J; Woolfson, DN.
Protein Science 10 668-673 (2001) (Abstract)
21 . Sticky-end Assembly of a Designed Peptide Fiber Provides Insight Into Protein Fibrillogenesis
Pandya, MJ; Spooner, GM; Sunde, M; Thorpe, JR; Rodger, A; Woolfson, DN.
Biochemistry 39 8728-8734 (2000) (Abstract)
20 . A Ligand-reversible Dimerization System For Controlling Protein-protein Interactions
Rollins, CT; Rivera, VM; Woolfson, DN; Keenan, T; Hatada, M; Adams, SE; Andrade, LJ; Yaeger, D; van Schravendijk MR; Holt, DA; Gilman, M; Clackson, T.
Proc. Natl. Acad. Sci. U.S.A. 97 (13) 7096-7101 (2000) (Abstract)
19 . Core-directed Protein Design. I. An Experimental Method For Selecting Stable Proteins from Combinatorial Libraries
Finucane, MD; Tuna, M; Lees, JH; Woolfson, DN.
Biochemistry 38 (36) 11613-11623 (1999) (Abstract)
18 . Core-directed Protein Design. II. Rescue of a Multiply Mutated and Destabilized Variant of Ubiquitin
Finucane, MD; Woolfson, DN.
Biochemistry 38 (36) 11604-11612 (1999 ) (Abstract)
17 . Regulation of Hsp90 ATPase Activity by Tetratricopeptide Repeat (TPR)-domain Co-chaperones
Prodromou, C; Siligardi, G; Obrien, R; Woolfson, DN; Regan, L; Panaretou, B; Ladbury, JE; Piper, PW; Pearl, LH.
EMBO Journal 18 (3) 754-762 (1999) (Abstract)
16 . Core-directed Protein Design: Selection of Stable Variants from Libraries of Hydrophobic Core Mutants
Finucane, MD; Lees, JH; Woolfson, DN.
FASEB Journal 11 (9 SS) 90 (1997) (No Abstract Available)
15 . Determinants of Strand Register in Antiparallel Beta-sheets of Proteins
Hutchinson, EG; Sessions, RB; Thornton, JM; Woolfson, DN.
Protein Science 7 (11) 2287-2300 (1998) (Abstract)
14 . Coiled-coil Assembly by Peptides With Non-heptad Sequence Motifs
Hicks, MR; Holberton, DV; Kowalczyk, C; Woolfson, DN.
Folding & Design 2 (3) 149-158 (1997) (Abstract)
13 . Buried Polar Residues and Structural Specificity in the GCN4 Leucine Zipper
Gonzalez, L; Woolfson, DN; Alber, T.
Nature Structural Biology 3 (12) 1011-1018 (1996) (Abstract)
12 . A Designed Heterotrimeric Coiled-coil
Nautiyal, S; Woolfson, DN; King, DS; Alber, T.
Biochemistry 34 (37) 11645-11651 (1995) (Abstract)
11 . Predicting Oligomerization States of Coiled Coils
Woolfson, DN; Alber, T.
Protein Science 4 (8) 1596-1607 (1995) (Abstract)
10 . Folding of Beta-structure in Proteins
Evans, PA; Gladwin, ST; Gopalakrishnan, B; Tisi, LC; Woolfson, DN; Murray, JH; Packman, LC; Trotter, BW; Mackay, JP; Williams, DH; Thornton, JM.
FASEB Journal 9 (6) A1239 (1995) (No Abstract Available)
9 . Dissecting the Structure of a Partially Folded Protein - Circular- Dichroism and Nuclear-magnetic-resonance Studies of Peptides from Ubiquitin
Cox, JPL; Evans, PA; Packman, LC; Williams, DH; Woolfson, DN.
Journal of Molecular Biology 234 (2) 483-492 (1993) (No Abstract Available)
8 . Topological and Stereochemical Restrictions in Beta-sandwich Protein Structures
Woolfson, DN; Evans, PA; Hutchinson, EG; Thornton, JM.
Protein Engineering 6 (5) 461-470 (1993) (Abstract)
7 . Protein Folding in the Absence of the Solvent Ordering Contribution to the Hydrophobic Interaction
Woolfson, DN; Cooper, A; Harding, MM; Williams, DH; Evans, PA.
Journal of Molecular Biology 229 (2) 502-511 (1993) (No Abstract Available)
6 . Depicting Topology and Handedness in Jellyroll Structures
Stirk, HJ; Woolfson, DN; Hutchinson, EG; Thornton, JM.
FEBS Letters 308 (1) 1-3 (1992) (Abstract)
5 . Conserved Positioning of Proline Residues in Membrane-spanning Helices of Ion-channel Proteins
Woolfson, DN; Mortishiresmith, RJ; Williams, DH.
Biochemical and Biophysical Research Communications 175 (3) 733-737 (1991) (No Abstract Available)
4 . Characterization of a Partially Denatured State of a Protein by 2- Dimensional NMR - Reduction of the Hydrophobic Interactions in Ubiquitin
Harding, MM; Williams, DH; Woolfson, DN.
Biochemistry 30 (12) 3120-3128 (1991) (Abstract)
3 . Hydrophobic Clustering in Nonnative States of a Protein - Interpretation of Chemical-shifts in NMR-spectra of Denatured States of Lysozyme
Evans, PA; Topping, KD; Woolfson, DN; Dobson, CM.
Proteins-structure Function and Genetics 9 (4) 248-266 (1991) (Abstract)
2 . A 3-disulfide Derivative of Hen Lysozyme - Structure, Dynamics and Stability
Radford, SE; Woolfson, DN; Martin, SR; Lowe, G; Dobson, CM.
Biochemical Journal 273 (Pt1) 211-217 (1991) (Abstract)
1 . The Influence of Proline Residues on Alpha-helical Structure
Woolfson, DN; Williams, DH.
FEBS Letters 277 (1-2) 185-188 (1990) (Abstract)

Abstracts

47 . ZiCo: A Peptide Designed to Switch Folded State upon Binding Zinc.
E Cerasoli, Sharpe BK, DN Woolfson
J. Am. Chem. Soc 127 15008-9 (2005)
We describe a novel approach to the design of a metal-triggered conformational switch. Specifically, two distinct protein-folding motifs were merged into one polypeptide sequence. The target structures were an alpha-helical coiled-coil trimer and zinc-bound monomer. Solution-phase spectroscopic, sedimentation, and binding studies confirmed the key aspects of the design. Both forms of the peptide were cooperatively folded, and the switch between them was reversible. This design process potentially presents a novel route to peptide-based biosensors.
46 . The design of coiled-coil structures and assemblies
DN Woolfson
Adv Protein Chem 70 79-112 (2005)
Protein design allows sequence-to-structure relationships in proteins to be examined and, potentially, new protein structures and functions to be made to order. To succeed, however, the protein-design process requires reliable rules that link protein sequence to structure?function. Although our present understanding of coiled-coil folding and assembly is not complete, through numerous bioinformatics and experimental studies there are now sufficient rules to allow confident design attempts of naturally observed and even novel coiled-coil motifs. This review summarizes the current design rules for coiled coils, and describes some of the key successful coiled-coil designs that have been created to date. The designs range from those for relatively straightforward, naturally observed structures-including parallel and antiparallel dimers, trimers and tetramers, all of which have been made as homomers and heteromers-to more exotic structures that expand the repertoire of Nature's coiled-coil structures. Examples in the second bracket include a probe that binds a cancer-associated coiled-coil protein; a tetramer with a right-handed supercoil; sticky-ended coiled coils that self-assemble to form fibers; coiled coils that switch conformational state; a three-component two-stranded coiled coil; and an antiparallel dimer that directs fragment complementation of larger proteins. Some of the more recent examples show an important development in the field; namely, new designs are being created with function as well as structure in mind. This will remain one of the key challenges in coiled-coil design in the next few years. Other challenges that lie ahead include the need to discover more rules for coiled-coil prediction and design, and to implement these in prediction and design algorithms. The considerable success of coiled-coil design so far bodes well for this, however. It is likely that these challenges will be met and surpassed.
45 . MaP peptides: Programming the self-assembly of peptide-based mesoscopic matrices
MG Ryadnov, DN Woolfson
J. Am. Chem. Soc 127 12407-12415 (2005)
We describe an approach that utilizes nonlinear peptides to direct the assembly of previously reported Self-Assembling Fibers (SAFs). The SAF system comprises two complementary linear peptides, SAF-p1 and SAF-p2a, which combine to form exclusively linear, nonbranched fibers. The Matrix-Programming (MaP) peptides described herein are based on these peptides: they comprise two or three half-peptide blocks derived from the SAF peptides, which are conjugated via dendritic hubs. Different MaP peptides coassembled with the standard SAF peptides to form specific structures, such as hyperbranched networks, polygonal matrices, and regularly segmented and terminated fibers. The role of each half-peptide block in dictating the different features has been elucidated. This provides a strong basis for designing new peptide-based nanostructured materials from the bottom up.
44 . Polar assembly in a designed protein fiber
AM Smith, SFA Acquah, N Bone, HW Kroto, MG Ryadnov, MSP Stevens, DRM Walton, & DN Woolfson*
Angew. Chem. Int. Ed. 44 325-328 (2005)
No Abstract
43 . Sequence and Structural Duality: Designing Peptides to Adopt Two Stable Conformations
MJ Pandya, E Cerasoli, A Joseph, RG, E Waite & DN Woolfson*
J. Am. Chem. Soc 126 17016-17024 (2004)
Abstract: To improve our understanding of conformational transitions in proteins, we are attempting the de novo design of peptides that switch structural state. Here, we describe coiled-coil peptides with sequence and structural duality; that is, features compatible with two different coiled-coil motifs superimposed within the same sequence. Specifically, we promoted a parallel leucine-zipper dimer under reducing conditions, and a monomeric helical hairpin in an intra-molecularly disulfide bridged state. Using an iterative process we engineered peptides that formed stable structures consistent with both targets under the different conditions. Finally for one of the designs, we demonstrated a one-way switch from the helical hairpin to the coiled-coil dimer upon addition of disulfide-reducing agents.
42 . Design and synthesis of a nitrogen-mustard derivative stabilised by apo-Neocarzinostatin
MD Urbaniak, JP Bingham, JA Hartley, DN Woolfson & S Caddick*
J. Med. Chem. 47 4710-4715 (2004)
Neocarzinostatin (NCS) is an antitumor antibiotic comprising a 1:1 protein-chromophore complex and exhibits cytotoxic action through DNA cleavage via H-abstraction. Cytotoxic activity resides with the chromophore 1 alone, while the protein (apoNCS) protects and transports labile 1. The naphthoate portion (2) of NCS chromophore (1) is important for binding to apoNCS and DNA intercalation. In this paper we describe our attempts to use apoNCS to improve the hydrolytic stability of novel bifunctional DNA alkylating agents. The nitrogen mustards, melphalan and chlorambucil, were both conjugated to 2, and the biological activities of these conjugates were assessed. Chlorambucil did not benefit from conjugation. The melphalan conjugate (6) formed covalent DNA adducts at guanine bases and exhibited greater in vitro cytotoxic activity than unmodified melphalan. Fluorescence and NMR spectroscopy showed that 6 binds to apoNCS. Binding to apoNCS-protected 6 reduced the extent of hydrolysis of the conjugate. This novel approach demonstrates for the first time that an enediyne apo-protein can be used to improve the stability of substances that are of potential interest in cancer chemotherapy.
41 . Biophysical and mutational analysis of the putative bZIP domain of Epstein-Barr virus EBNA 3C.
MJ West*, HM Webb, AJ Sinclair & DN Woolfson
J Virology 78 9431-9445 (2004)
Epstein-Barr virus nuclear antigen 3C (EBNA 3C) is essential for B-cell immortalization and functions as a regulator of viral and cellular transcription. EBNA 3C contains glutamine-rich and proline-rich domains and a region in the N terminus consisting of a stretch of basic residues followed by a run of leucine residues spaced seven amino acids apart. This N-terminal domain is widely believed to represent a leucine zipper dimerization motif (bZIP). We have performed the first structural and functional analysis of this motif and demonstrated that this domain is not capable of forming stable homodimers. Peptides encompassing the EBNA 3C zipper domain are approximately 54 to 67% alpha-helical in solution but cannot form dimers at physiologically relevant concentrations. Moreover, the EBNA 3C leucine zipper cannot functionally substitute for another homodimerizing zipper domain in domain-swapping experiments. Our data indicate, however, that the EBNA 3C zipper domain behaves as an atypical bZIP domain and is capable of self-associating to form higher-order alpha-helical oligomers. Using directed mutagenesis, we also identified a new role for the bZIP domain in maintaining the interaction between EBNA 3C and RBP-JK in vivo. Disruption of the helical nature of the zipper domain by the introduction of proline residues reduces the ability of EBNA 3C to inhibit EBNA 2 activation and interact with RBP-JK in vivo by 50%, and perturbation of the charge on the basic region completely abolishes this function of EBNA 3C.
40 . Fiber Recruiting (FiRe) Peptides: Non-Covalent Decoration of an Engineered Protein Scaffold
MG Ryadnov and DN Woolfson*
J Am Chem Soc 126 7454-7455 (2004)
No Abstract
39 . Engineered and Designed Peptide-based Fibrous Biomaterials
CE MacPhee & DN Woolfson*
Current Opinion in Solid State and Material Science 8 2 141-149 (2004)
Our increasing understanding of peptide and protein folding and assembly raises new possibilities for engineering novel self-assembling supramolecular structures and bio-inspired materials. Here we focus on peptides designed de novo, and natural systems that have been engineered to form extended protein fibres. Potential applications of such assemblies include the preparation of functionalised biomaterials for the development of new diagnostic devices, scaffolds to recruit cells for cell/tissue engineering, and templates for the assembly of inorganic materials.
38 . Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin
CG Benitez-Cardoza, K Stott, M Hirshberg, DN Woolfson & SE Jackson*
Biochemistry 43 5195-5203 (2004)
The stability, dynamic, and structural properties of ubiquitin and two Multiple hydrophobic core mutants were studied. One of the mutants (U4) has seven substitutions in the hydrophobic core (M1L, I3L, V5I, I3F, L15V. V17M, and V26L). On average, its side chains are larger than the wild-type, and it can thus be thought of as having an overpacked core. The other mutant (U7) has two substitutions (I3V and I13V). On average, it has smaller side chains than the wild-type, and it can therefore be considered to be underpacked. The three proteins are well- folded and show similar backbone dynamics (T-1, T-2, and HNOE values), indicating that the regular secondary structure extends over the same residue ranges. The crystallographic structure of U4 was determined. The final R-factor and R-free are 0.198 and 0.248. respectively, at 2.18 Angstrom resolution. The structure of U4 is very similar to wild-type ubiquitin. Remarkably. there are almost no changes in the positions of the C-alpha atoms along the entire backbone, and the hydrogen- bonding network is maintained. The mutations of the hydrophobic core are accommodated by small movements of side chains in the core of mutated and nonmutated residues. Unfolding and refolding kinetic studies revealed that U4 unfolds with the highest rates; however, its refolding rate constants are very similar to those of the wild-type protein. Conversely, U7 seems to be the most destabilized protein; its refolding rate constant is smaller than the other two proteins. This was confirmed by stopped-flow techniques and by H/D exchange methodologies. This work illustrates the possibility of repacking the hydrophobic core of small proteins and has important implications in the de novo design of stable proteins.
37 . Extended knobs-into-holes packing in classical and complex coiled-coil assemblies
John Walshaw and Derek N. Woolfson
Journal of Structural Biology 144 3 349-361 (2003)
This year marks the 50th anniversary of Crick's seminal paper on the packing of a-helices into coiled-coil structures. The central tenet of Crick's work is the interdigitation of side chains, which directs the helix-helix interactions; so called knobs-into-holes packing. Subsequent determinations of coiled-coil-protein sequences and structures confirmed the key features of Crick's model and established it as a fundamental concept in structural biology. Recently, we developed a program, SOCKET, to recognise knobs-into-holes packing in protein structures, which we applied to the Protein Data Bank to compile a database of coiled-coil structures. In addition to classic structures, the database reveals 4-helix bundles and larger helical assemblies. Here, we describe how the more-complex structures can be understood by extending Crick's principles for classic coiled coils. In the simplest case, each helix of a 2-stranded structure contributes a single seam of (core) knobs-into-holes to the helical interface. 3-, 4-, and 5-Stranded structures, however, are best considered as rings of helices with cycles of knobs-into-holes. These higher-order oligomers make additional (peripheral) knobs-into-holes that broaden the helical contacts. Combinations of core and peripheral knobs may be assigned to different sequence repeats offset within the same helix. Such multiple repeats lead to multi-faceted helices, which explain structures above dimers. For instance, coiled-coil oligomer state correlates with the offset of the different repeats along a sequence. In addition, certain multi-helix assemblies can be considered as conjoined coiled coils in which multi-faceted helices participate in more than one coiled-coil motif.
36 . "Belt and Braces": A Peptide-Based Linker System of de Novo Design
MG Ryadnov, B Ceyhan, CM Niemeyer & DN Woolfson
J Amer Chem Soc 125 31 9388-9394 (2003)
A new self-assembling peptide-based linker is described. The system comprises three leucine-zipper sequences of de novo design: one peptide, "the belt", templates the co-assembly of the other two half-sized peptides, "the braces". These basic features were confirmed by circular dichroism spectroscopy and analytical ultracentrifugation: when mixed the three peptides reversibly formed a predominantly helical and stable 1:1:1 ternary complex. Surface plasmon resonance experiments demonstrated assembly of the complex on gold surfaces, whilst the ability of the system to bring together peptide-bound cargo was demonstrated using colloidal gold nanoparticles. In the latter experiments, the nanoparticles were derivatized with the brace peptides prior to the addition of the belt. Transmission electron microscopy images of the resulting networks revealed regular 7 nm separations between adjacent particles, consistent with the 42-amino acid helical design of the belt and braces. To our knowledge, belt and braces is a novel concept in leucine-zipper assembly, and the first example of employing peptides to guide nanoparticle assembly.
35 . Introducing Branches into a Self-Assembling Peptide Fiber
MG Ryadnov & DN Woolfson
Angew. Chem. - Int. Ed. 42 3021-3023 (2003)
There is a growing interest in employing peptides as building blocks in the self-assembly of supramolecular structures.[1] Such assemblies have potential as novel scaffolds for functionalizing surfaces and in tissue engineering.[2] Elsewhere,[3] we describe a self-assembling fiber (SAF) system comprising two designed peptides (1 & 2, aka SAF-p1 and SAF-2a, Figure 1). These sequences are based on established design principles for leucine-zipper motifs.[4] However, unlike all other leucine zippers, which are blunt ended, the SAF peptides are designed to assemble with sticky ends that facilitate fibrillogenesis (Figure 1, Figure S1). Like other systems,[1a,c,f] the SAF peptides are linear and form exclusively linear and non-branching fibers when mixed; n.b. 2 is a slight redesign of the previously described SAF-p2,[3] this new design combines with 1 to give more-stable and better-ordered linear fibers (Figure S2A-D, supporting information). Natural protein fibers such as those formed by actin, collagen and fibrin branch. We set out to design special units to complement the standard SAF building blocks and, so, engineer branched fibers de novo. Here we report T-shaped peptides, T-SAFs, that co-assemble with the standard SAF peptides to give branched self-assembling fibers. In T-SAFs the ?bar? of the ?T? is the complete SAF peptide 2 and the ?stem? (peptide 3) is the N-terminal half of 2; the stem and the bar are joined via a linker, typically of three b-alanine (bAla) units, between the C-terminus of peptide 3 and the e-amino group of the central lysine (lysine-14) of peptide 2 (Figure 1). In principle, when combined with peptides 1 and 2, T-SAFs should promote assembly of orthogonally conjoined fibers.
34 . Engineering the morphology of a self-assembling protein fibre
MG Ryadnov & DN Woolfson
Nature Materials 2 5 329-332 (2003)
Biological assemblies provide inspiration for the development of new materials for a variety of applications. Our ability to realize this potential, however, is hampered by difficulties in producing and engineering natural biomaterials, and in designing them from new. We previously described a self-assembling system comprising two short complementary segments of straight synthetic polypeptides (dubbed standards in this report). Their interaction results in the formation of long fibres ? about 50 nm in diameter ? that extend straight and without branching for tens to hundreds of micrometres. Our aim is to influence and, ultimately, to control fibre morphology. Here, we show that the standard peptides can be supplemented with special peptides to effect morphological changes in the fibres. Specifically, we created half-sized subunits of the standard peptides, which were combined to make nonlinear peptides. When mixed with the standard peptides, these nonlinear peptides produced kinked, waved and branched fibres. We related the numbers of these features to the special/standard ratios empirically. Furthermore, the extent and frequency of kinking was altered by changing the standard-fibre background: more kinking was observed in a background of thinner, less-stable fibres. The ability to perform such transformations holds promise for bottom-up assembly and engineering-responsive mimetic materials for applications in surface and tissue engineering.
33 . Chemical Synthesis and Cytotoxicity of Dihydroxylated Cyclopentenone Analogues of Neocarzinostatin Chromophore
MD Urbaniak, LM Frost, JP Bingham, LR Kelland, JA Hartley, DN Woolfson & S Caddick
Bioorganic & Medicinal Chemistry Letters 13 12 329-332 (2003)
Compounds containing the naphthoate moiety of Neocarzinostatin chromophore or 2-hydroxynaphthoate have been synthesized and evaluated for cytotoxic activity against a leukemia cell line and a small panel of human-tumor cell lines. Those compounds containing a cyclopentenone moiety were active, with the carbonyl group being essential for biological activity.
32 . Solution Structure of a Novel Chromoprotein Derived from Apo-Neocarzinostatin and a Synthetic Chromophore.
Michael D. Urbaniak, Frederick W. Muskett, Michael D. Finucane, Stephen Caddick and Derek N. Woolfson
Biochemistry 41 11731-11739 (2002)
The natural complex Neocarzinostatin comprises a labile chromophore non-covalently bound to an 11.2 kDa protein. We present the first high-resolution structure of a novel complex derived from the recombinant apo-protein bound to a non-natural synthetic chromophore. Fluorescence and nuclear magnetic resonance spectroscopy were used to probe the strength and location of binding. Binding occurred in a similar location to the natural chromophore, but with a distinct orientation. These results provide structural evidence that the apo-protein can readily accommodate small drug-like entities, other than the natural chromophore within its binding cleft. The clinical use of the natural complex to act as a drug-delivery system described by others, together with the promiscuity in binding reported here, suggests potential applications for small-molecule binding by apo-Neocarzinostatin.
31 . Mini-proteins Trp the light fantastic
Gellman SH, Woolfson DN
Nature Structural Biology 9 6 408-410 (2002)
A new 20-residue peptide represents the smallest example to date of cooperatively folded tertiary structure. This achievement provides a new tool for elucidating protein conformational preferences. The mini-protein should serve as a fruitful platform for protein design.
30 . Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37
Siligardi, G; Panaretou, B; Meyer, P; Singh, S; Woolfson, DN; Piper, PW; Pearl, LH; Prodromou, C.
J. Biol. Chem. 277 23 20151-20159 (2002)
In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle, and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a 'scaffold', recruiting Hsp70 and the bound client to Hsp90 early in the cycle, and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialised co-chaperone, Cdc37p/p50cdc37 whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50cdc37 like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a 'relaxed' ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50cdc37 binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50cdc37 is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50cdc37 forms a stable complex with geldanamycin-bound Hsp90, and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.
29 . Generalised Crick equations for modelling non-canonical coiled coils
Offer, G; Hicks, MR; Woolfson, DN;
J. Struct. Biol. 137 41-53 (2002)
Crick envisaged the a-helical coiled coil to result from systematic bending of an a-helix such that every seventh residue was structurally equivalent and derived equations for the coordinates of the backbone atoms. Crick¹s predictions were vindicated experimentally and coiled-coil sequences shown to have hydrophobic residues alternately spaced 3 and 4 residues apart. Nonetheless, in some coiled coils such canonical heptad repeats are interrupted by inserts of 3 or 4 residues generating decad and hendecad motifs. The supercoiling of such coiled coils varies with the sequence pattern, being left or right-handed in purely heptad-based or hendecad-based motifs, respectively. To model such structures, we describe how the Crick equations can be extended to coiled coils with a mixture of motifs where the pitch is not constant. Using the analogy of the bending of a beam, we took the tilt angle to change linearly along the major helix and the pitch of a motif to be affected by neighboring motifs depending on the rigidity of the a-helical strands. We tested our approach by fitting the two, three and four-stranded non-canonical coiled coils of GrpE, haemagglutinhemagglutinin and tetrabrachion. The backbone atoms of the model and crystal structures agreed with rms deviations of <1.1 Angstroms.
28 . Investigating the tolerance of coiled-coil peptides to non-heptad sequence inserts
Hicks, MR; Walshaw, J; Woolfson, DN;
J. Struct. Biol. 137 73-81 (2002)
Coiled-coil motifs foster a wide variety of protein-protein interactions. Canonical coiled coils are based on seven-residue repeats, which guide the folding and assembly of amphipathic a-helices. In many cases such repeats remain unbroken for tens to hundreds of residues. However, the sequences of an increasing number of putative and characterised coiled coils digress from this pattern. We probed the consequences of non-heptad inserts using a designed leucine-zipper system. The parent peptide, SKIP0, which had four contiguous heptads, was confirmed as a parallel homodimer by circular dichroism spectroscopy and analytical ultracentrifugation. Seven daughter peptides were constructed in which one to seven alanine residues were inserted between the central heptads of SKIP0. Like SKIP0, SKIP7 formed a stable helical dimer, but the other peptides were highly destabilised, with the order of dimer stability SKIP4 >> SKIP5 > SKIP6 > SKIP3 > SKIP2 > SKIP1. These results are consistent with an extended theory of coiled-coil assembly in which coiled-coil-compatible motifs are based on three- and four-residue spacings, and most notably heptad (seven-residue) and hendecad (eleven-residue) repeats. Thus, they help explain why in natural sequences, inserts after canonical heptad repeats most commonly of four-residues. Possible biological roles for non-heptad inserts are discussed.
27 . A designed system for assessing how sequence affects alpha-to-beta conformational transitions in proteins
Ciani, B; Hutchinson, EG; Sessions, RB; Woolfson, DN.
J. Biol. Chem. 277 10150-10155 (2002)
The role of amino-acid sequence in conformational switching observed in prions and proteins associated with amyloid diseases is not well understood. To study a-to-b conformational transitions, we designed a series of peptides with structural duality; namely, peptides with sequence features of both an a-helical leucine zipper and a b-hairpin. The parent peptide, Template-a, was designed to be a canonical leucine-zipper motif and was confirmed as such using circular dichroism spectroscopy and analytical ultracentrifugation. To introduce b-structure character into the peptide, glutamine residues at sites away from the leucine-zipper dimer interface were replaced by threonine to give Template-aT. Unlike the parent peptide, Template-aT underwent a heat-inducible switch to b-structure, which reversibly formed gels containing amyloid-like fibrils. In contrast to certain other natural proteins where destabilisation of the native states facilitate transitions to amyloid, destabilisation of the leucine-zipper form of Template-aT did not promote a transformation. Cross-linking the termini of the peptides compatible with the alternative b-hairpin design, however, did promote the change. Furthermore, despite screening various conditions, only the internally cross-linked form of the parent, Template-a, peptide formed amyloid-like fibrils. These findings demonstrate that, in addition to general properties of the polypeptide backbone, specific residue placements that favour b-structure promote amyloid formation.
26 . Core-directed Protein Design
Woolfson, DN.
Curr. Opin. Struct. Biol. 11 464-471 (2001)
For various reasons, it seems sensible to redesign or design proteins from the inside out. Past approaches in this field have involved iterations of mutagenesis and characterisation to 'evolve' designs. Increasingly, combinatorial approaches are being taken to select 'fit' sequences from libraries of variant proteins. In particular, in silico methods have been used to good effect. More recently, experimental methods have been developed and improved. We are now in a position to redesign stability and function into natural protein frameworks confidently and to attempt de novo designs for more ambitious targets.
25 . Guidelines For the Assembly of Novel Coiled-coil Structures: Alpha-sheets and Alpha-cylinders.
Walshaw, J; Shipway, JM; Woolfson, DN..
Biochem Soc Symposium 68, "From Protein Folding to New Enzymes" Eds. A.Berry & SE Radford 111-123 (2001)
The coiled coil is a ubiquitous motif that guides many different protein-protein interactions. The accepted hallmark of coiled coils is a 7-residue (heptad) sequence repeat. With positions of this repeat labelled a-b-c-d-e-f-g residues at a and d tend to be hydrophobic. Such sequences form amphipathic alpha-helices, which assemble into helical bundles via knobs-into-holes (KIH) interdigitation of residues from neighbouring helices. We wrote an algorithm, SOCKET, to identify this packing in protein structures, and used this to gather a database of coiled-coil structures from the Protein Data Bank (PDB). Surprisingly, in addition to commonly accepted structures with a single, contiguous heptad repeat, we identified sequences with multiple, offset heptad repeats. These 'new' sequence patterns help explain oligomer-state specification in coiled coils. Here we focus on the structural consequences for sequences with two heptad repeats offset by two residues; i.e. a/f'-b/g'-c/a'-d/b'-e/c'-f/d'-g/e'. This sets up two hydrophobic seams on opposite sides of the helix formed. We describe how such helices may combine to bury these hydrophobic surfaces in two different ways and form two distinct structures: open "alpha-sheets" and closed "alpha-cylinders". We highlight these with descriptions of natural structures and outline possibilities for protein design.
24 . Biophysical Analysis of Natural Variants of the Multimerization Region of Epstein-Barr Virus Lytic-switch Protein Bzlf1
Hicks, MR; Balesaria, S; Medina-Palazon, C; Pandya, MJ; Woolfson, DN; Sinclair, AJ.
Journal of Virology 75 5381-5384 (2001)
BZLF1 plays a key role in the induction of Epstein-Barr virus (EBV) replication. On the basis of limited sequence homology and mutagenesis experiments, BZLF1 has been described as a member of the bZip family of transcription factors, but this prospect has not been rigorously tested to date. Here, we present biophysical analysis of the multimerization domain of BZLF1, from three natural variants of EBV, and demonstrate for the first time that the region between amino acids 196 and 227 is sufficient to direct folding as a coiled-coil dimer in vitro.
23 . Socket: a Program For Identifying and Analysing Coiled-coil Motifs Within Protein Structures
Walshaw, J; Woolfson, DN.
Journal of Molecular Biology 307 1427-1450 (2001)
The coiled coil is arguably the simplest protein-structure motif and probably the most ubiquitous facilitator of protein-protein interactions. Coiled coils comprise two or more a-helices that wind around each other to form supercoils. The hallmark of most coiled coils is a regular sequence pattern known as the heptad repeat. Despite this apparent simplicity and relatedness at the sequence level, coiled coils display a considerable degree of structural diversity: the helices may be arranged parallel or anti-parallel and may form a variety of oligomer states. To aid studies of coiled coils, we developed SOCKET, a computer program to identify these motifs automatically in protein structures. We used SOCKET to gather a set of unambiguous coiled-coil structures from the Protein Data Bank (PDB). Rather than searching for sequence features, the algorithm recognises the characteristic knobs-into-holes side-chain packing of coiled coils; this proved to be straightforward to implement and was able to distinguish coiled coils from the great majority of helix-helix packing arrangements observed in globular domains. SOCKET unambiguously defines coiled-coil helix boundaries, oligomerisation states and helix orientations, and also assigns heptad registers. Structures retrieved from the PDB included parallel and anti-parallel variants of 2-, 3- and 4-stranded coiled coils, one example of a parallel pentamer and a small number of structures that extend the classical description of a coiled-coil. We anticipate that our structural database and the associated sequence data that we have gathered will be of use in identifying principles for coiled-coil assembly, prediction and design. To illustrate this we give examples of sequence and structural analyses of the structures that are possible using the new data bases, and we present amino-acid profiles for the heptad repeats of different motifs.
22 . Open-and-shut Cases in Coiled-coil Assembly: Alpha-sheets and Alpha-cylinders
Walshaw, J; Woolfson, DN.
Protein Science 10 668-673 (2001)
The coiled coil is a ubiquitous protein-folding motif. It is generally accepted that coiled coils are characterised by sequence patterns known as heptad repeats. Such patterns direct the formation and assembly of amphipathic alpha-helices, the hydrophobic faces of which interface in a specific manner first proposed by Crick and termed knobs-into-holes packing. We developed software, SOCKET, to recognise this packing in protein structures. As expected, in a trawl of the protein data bank we found examples of canonical coiled coils with a single contiguous heptad repeat. In addition, however, we identified structures with multiple, overlapping heptad repeats. This observation extends Crick's original postulate: multiple, offset heptad repeats help explain assemblies with more than two helices. Indeed, we have found that the sequence offset of the multiple heptad repeats is related to coiled-coil oligomer state. Here we focus on one particular sequence motif in which two heptad repeats are offset by two residues. This offset sets up two hydrophobic faces separated by 150-160 degrees around the alpha-helix. In turn, two different combinations of these faces are possible. Either similar or opposite faces can interface, which leads to open or closed multi-helix assemblies. Accordingly, we refer to these two forms as alpha-sheets and alpha-cylinders. We illustrate these structures with our own predictions and by reference to natural variants on these designs that have recently come to light.
21 . Sticky-end Assembly of a Designed Peptide Fiber Provides Insight Into Protein Fibrillogenesis
Pandya, MJ; Spooner, GM; Sunde, M; Thorpe, JR; Rodger, A; Woolfson, DN.
Biochemistry 39 8728-8734 (2000)
Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had "sticky-ends" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with "blunt-ends" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. Furthermore, X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred microns long and twenty times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.
20 . A Ligand-reversible Dimerization System For Controlling Protein-protein Interactions
Rollins, CT; Rivera, VM; Woolfson, DN; Keenan, T; Hatada, M; Adams, SE; Andrade, LJ; Yaeger, D; van Schravedijk MR; Holt, DA; Gilman, M; Clackson, T.
Proc. Natl. Acad. Sci. U.S.A. 97 (13) 7096-7101 (2000)
Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligand-binding site (Phe-36 --> Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (FM) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a "reverse dimerization" regulatory system involving FM fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated FM domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.
19 . Core-directed Protein Design. I. An Experimental Method For Selecting Stable Proteins from Combinatorial Libraries
Finucane, MD; Tuna, M; Lees, JH; Woolfson, DN.
Biochemistry 38 (36) 11613-11623 (1999)
The design of proteins represents a significant challenge to modern-day structural biology. A major obstacle here is the specification of well-packed hydrophobic cores to drive folding and stabilisation of the target. Computational approaches have been used to alleviate this by testing alternate sequences prior to the production and characterisation of a few proteins. Here we present the experimental counterpart of this approach. We selected stable variants from a library of ubiquitin hydrophobic-core mutants as follows: hexahistidine-tagged proteins were displayed on the surface of phage; these protein-phage were immobilised onto Ni1-coated surfaces; the bound fusion-phage were treated with protease to remove unstable or poorly folded proteins; stable phage fusions were eluted and infected into E. coli, which allowed amplification for further selection, sequencing, or protein expression. Two Ni-derivatised supports were tested: Ni-NTA chips for surface plasmon resonance (SPR), and Ni-NTA agarose beads. SPR carried the advantage that the selection process could be monitored directly. This allowed individual clones and experimental conditions to be tested rapidly prior to preparative panning of the library, which was done using Ni-NTA agarose beads. We demonstrate the method by selecting stable core mutants of ubiquitin, the characterisation of which is described in the accompanying paper (Finucane & Woolfson, accompanying paper). As our method selects based only on structure and stability, it will be of use in improving the stabilities and structural specificities of proteins of de novo design, and in establishing rules that link sequence and structure.
18 . Core-directed Protein Design. II. Rescue of a Multiply Mutated and Destabilized Variant of Ubiquitin
Finucane, MD; Woolfson, DN.
Biochemistry 38 (36) 11604-11612 (1999 )
We have applied the method described in the accompanying paper (Finucane et al., accompanying paper), namely stability-based selection using phage display, to explore the sequence requirements for packing in the hydrophobic core of ubiquitin. In contrast to the parent protein, which was a structurally compromised mutant, the selected variants could be overexpressed and purified in yields for structural studies. In particular, CD and NMR measurements showed that the selectants folded correctly to stable native-like structures. These points demonstrate the utility of our core-directed method for stabilising and redesigning proteins. In addition and in contrast to foregoing studies on other proteins, which suggest that hydrophobic cores permit substitutions provided that hydrophobicity and core volumes are generally conserved, we find that the core of ubiquitin is surprisingly intolerant of amino-acid substitutions; variants that survived our selection showed a clear consensus for the wild-type sequence. It is probable that our results differed from those from other groups for two reasons: first, ubiquitin may be unusual in that it has strict sequence requirements for its structure and stability. We discuss this result in light of sequence conservation in the eukaryotic ubiquitins and proteins of the ubiquitin structural superfamily. Second, our mutants were selected solely on the basis of stability, in contrast to the other studies that rely on function-based selection. The latter may lead to proteins that are more plastic and tolerant of substitutions.
17 . Regulation of Hsp90 ATPase Activity by Tetratricopeptide Repeat (TPR)-domain Co-chaperones
Prodromou, C; Siligardi, G; Obrien, R; Woolfson, DN; Regan, L; Panaretou, B; Ladbury, JE; Piper, PW; Pearl, LH.
EMBO Journal 18 (3) 754-762 (1999)
The in vivo function of the heat shock protein 90 (Hsp90) molecular chaperone is dependent on the binding and hydrolysis of ATP, and on interactions with a variety of co-chaperones containing tetratricopeptide repeat (TPR) domains. We have now analysed the interaction of the yeast TPR-domain co-chaperones Sti1 and Cpr6 crith yeast Hsp90 by isothermal titration calorimetry, circular dichroism spectroscopy and analytical ultracentrifugation, and determined the effect of their binding on the inherent ATPase activity of Hsp90, Sti1 and Cpr6 both bind with sub-micromolar affinity, with Sti1 binding accompanied by a large conformational change. Two co- chaperone molecules bind per Hsp90 dimer, and Sti1 itself is found to be a dimer in free solution. The inherent ATPase activity of Hsp90 is completely inhibited by binding of Sti1, but is not affected by Cpr6, although Cpr6 can reactivate the ATPase activity by displacing Sti1 from Hsp90, Bound Sti1 makes direct contact with, and blocks access to the ATP-binding site in the N-terminal domain of Hsp90, These results reveal an important role for TPR-domain co-chaperones as regulators of the ATPase activity of Hsp90, showing that the ATP- dependent step in Hsp90-mediated protein folding occurs after the binding of the folding client protein, and suggesting that ATP hydrolysis triggers client-protein release.
16 . Core-directed Protein Design: Selection of Stable Variants from Libraries of Hydrophobic Core Mutants
Finucane, MD; Lees, JH; Woolfson, DN.
FASEB Journal 11 (9 SS) 90 (1997)

(No Abstract Available)

15 . Determinants of Strand Register in Antiparallel Beta-sheets of Proteins
Hutchinson, EG; Sessions, RB; Thornton, JM; Woolfson, DN.
Protein Science 7 (11) 2287-2300 (1998)
Antiparallel beta-sheets present two distinct environments to inter- strand residue pairs: beta(A,HB) sites have two backbone hydrogen bands; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only similar to 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic- Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi(1) conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi(1) geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche(-) conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de nova design. Our findings are discussed in light of previous analyses and experimental studies.
14 . Coiled-coil Assembly by Peptides With Non-heptad Sequence Motifs
Hicks, MR; Holberton, DV; Kowalczyk, C; Woolfson, DN.
Folding & Design 2 (3) 149-158 (1997)
Background: The seven-residue heptad repeat is the accepted hallmark of coiled coils. In extended filamentous proteins, however, contiguous patterns of heptads are often disrupted by 'skips' and 'stammers'. The structural consequences and roles of these digressions are not understood. Results: In a cytoskeleton protein from Giardia lamblia, heptads flank eleven-residue units (hendecads) to give a 7-11-7 motif that dominates the sequence. Synthetic peptides made to the consensus sequence of this motif fold in solution to fully helical, parallel dimers. Both the sequence pattern and these experimental data are consistent with the coiled-coil model, We note that breaks in other extended coiled coils can also be reconciled by hendecad insertions, Conclusions: The heptad paradigm for the coiled coil must be expanded to include hendecads. As different combinations of heptads and hendecads will give different overall sequence motifs, we propose that these provide a mechanism to promote cognate protein pairings during the folding of extended coiled coils in the cell.
13 . Buried Polar Residues and Structural Specificity in the GCN4 Leucine Zipper
Gonzalez, L; Woolfson, DN; Alber, T.
Nature Structural Biology 3 (12) 1011-1018 (1996)
A conserved asparagine (Asn 16) buried in the interface of the GCN4 leucine zipper selectively favours the parallel, dimeric, coiled-coil structure. To test if other polar residues confer oligomerization specificity, the structural effects of Gln and Lys substitutions for Asn 16 were characterized. Like the wild-type peptide, the Asn16Lys mutant formed exclusively dimers. In contrast, Gln 16, despite its chemical similarity to Asn, allowed the peptide to form both dimers and trimers. The Gln 16 side chain was accommodated by qualitatively different interactions in the dimer and trimer crystal structures. These findings demonstrate that the structural selectivity of polar residues results not only from the burial of polar atoms, but also depends on the complementarity of the side-chain stereochemistry with the surrounding structural environment.
12 . A Designed Heterotrimeric Coiled-coil
Nautiyal, S; Woolfson, DN; King, DS; Alber, T.
Biochemistry 34 (37) 11645-11651 (1995)
Principles that guide folding of coiled coils were tested by designing three peptides that preferentially associate with each other to form a heterotrimeric coiled coil. The core positions of the designed helices contained residues that promote formation of trimeric coiled coils. Ionic interactions were employed to mediate heterospecificity, and negative design was used to favor formation of the heterotrimer over alternative arrangements. A program was written to select sequences that maximized the number of attractive interhelical interactions in a parallel heterotrimer and the number of repulsive electrostatic interactions in alternative species. Solution studies indicate that an equimolar mixture of the three peptides forms a helical trimer with high specificity and stability. These results validate the principles used to guide the design and suggest that the heterotrimer may serve as a useful, autonomous trimerization domain.
11 . Predicting Oligomerization States of Coiled Coils
Woolfson, DN; Alber, T.
Protein Science 4 (8) 1596-1607 (1995)
An algorithm based on the profile method was developed that faithfully distinguishes between the amino acid sequences of dimeric and trimeric coiled coils. Normalized sequence profiles derived from nonhomologous, two- and three-stranded, coiled-coil sequences with unambiguous registers were used to assign dimer and trimer propensities to test sequences. The difference between the dimer and trimer profile scores accurately reflected the preferred oligomerization state. The method relied on two strategies that may be generally applicable to profile calculations-profile values of solvent-exposed residues and of amino acids that were underrepresented in the database were given zero weight. Differences between the dimer and trimer profiles revealed sequence patterns that match and extend experimental studies of oligomer specification.
10 . Folding of Beta-structure in Proteins
Evans, PA; Gladwin, ST; Gopalakrishnan, B; Tisi, LC; Woolfson, DN; Murray, JH; Packman, LC; Trotter, BW; Mackay, JP; Williams, DH; Thornton, JM.
FASEB Journal 9 (6) A1239 (1995)

(No Abstract Available)

9 . Dissecting the Structure of a Partially Folded Protein - Circular- Dichroism and Nuclear-magnetic-resonance Studies of Peptides from Ubiquitin
Cox, JPL; Evans, PA; Packman, LC; Williams, DH; Woolfson, DN.
Journal of Molecular Biology 234 (2) 483-492 (1993)

(No Abstract Available)

8 . Topological and Stereochemical Restrictions in Beta-sandwich Protein Structures
Woolfson, DN; Evans, PA; Hutchinson, EG; Thornton, JM.
Protein Engineering 6 (5) 461-470 (1993)
Chain topology in beta-structured protein domains and handedness associated with it are discussed. Previously, other workers have shown that by considering just two restrictions-structures that are left-handed and/or have loops that cross can be disregarded-the number of topologies associated with such structures is expected to be severely limited. By way of example, we determine the number of topologies compatible with a six-stranded antiparallel beta-sandwich. Without restriction on the type of strand - strand connection allowed but with elimination of symmetry related structures 360 topologies are possible. If connections between parallel strands are disqualified the number is reduced, 10-fold, to 36. The figure is cut to 24 when structures with loop crossings are eliminated. Handedness in these structures is examined in detail and from this a rationale for the observed predominance of right-handed forms of beta- structures is presented. The 24 structures can be considered as a set of right- and left-handed pairs of 12 topologies. All but two of these pairs can be assigned hands on the basis of existing rules. Six of the structures are found to occur in the Brookhaven Protein Databank and all are right-handed. This study provides a basis for protein design projects which might, for example, attempt the synthesis of unobserved protein topologies. Of the 24 structures in the final set eight are examples of the classic Greek key fold. Thus, the predominance of this motif among all-beta proteins can be attributed in part to these topological constraints. The possible physicochemical origins of the structural selection rules and additional factors which might contribute to the particular favourability of certain structures are also explored.
7 . Protein Folding in the Absence of the Solvent Ordering Contribution to the Hydrophobic Interaction
Woolfson, DN; Cooper, A; Harding, MM; Williams, DH; Evans, PA.
Journal of Molecular Biology 229 (2) 502-511 (1993)

(No Abstract Available)

6 . Depicting Topology and Handedness in Jellyroll Structures
Stirk, HJ; Woolfson, DN; Hutchinson, EG; Thornton, JM.
FEBS Letters 308 (1) 1-3 (1992)
The jellyroll structure is a special case of the Greek key topology and, to date, has only been observed in complete form in one of its four possible arrangements. Like other elements of super-secondary structure involving the beta-strand (e.g. the beta-alpha-beta unit) the known structure forms a right-handed superhelix. The possibility of losing such tertiary information and other problems associated with representing these structures by two-dimensional topology diagrams are discussed. A series of rules are presented which allow this three-dimensional information to be represented in two- dimensional topology diagrams from which the handedness of a jellyroll structure can be determined.
5 . Conserved Positioning of Proline Residues in Membrane-spanning Helices of Ion-channel Proteins
Woolfson, DN; Mortishiresmith, RJ; Williams, DH.
Biochemical and Biophysical Research Communications 175 (3) 733-737 (1991)

(No Abstract Available)

4 . Characterization of a Partially Denatured State of a Protein by 2- Dimensional NMR - Reduction of the Hydrophobic Interactions in Ubiquitin
Harding, MM; Williams, DH; Woolfson, DN.
Biochemistry 30 (12) 3120-3128 (1991)
A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta- sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha- helix in the N-state. There is evidence for some fixed side-chain- side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.
3 . Hydrophobic Clustering in Nonnative States of a Protein - Interpretation of Chemical-shifts in NMR-spectra of Denatured States of Lysozyme
Evans, PA; Topping, KD; Woolfson, DN; Dobson, CM.
Proteins-structure Function and Genetics 9 (4) 248-266 (1991)
Chemical shifts of resonances of specific protons in the H-1 NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded "molten globule" state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.
2 . A 3-disulfide Derivative of Hen Lysozyme - Structure, Dynamics and Stability
Radford, SE; Woolfson, DN; Martin, SR; Lowe, G; Dobson, CM.
Biochemical Journal 273 (Pt1) 211-217 (1991)
A three-disulphide derivative of hen egg-white lysozyme was made by selective reduction and carboxymethylation of one of the four original disulphide bridges. N-Terminal sequencing and two- dimensional H-1-n.m.r. spectroscopy revealed that the disulphide bridge linking cysteine residues 6 and 127 had been modified and that the three remaining disulphide bonds were native-like in nature. Analysis of COSY and NOESY spectra indicated that the three- disulphide lysozyme (CM6,127-lysozyme retains the same secondary and tertiary structure as its four-disulphide counterpart; its stability to pH and temperature is, however, dramatically decreased. N.m.r. spectroscopy was used to characterize the thermal folding and unfolding transition of CM6,127-lysozyme. Not only is the transition still a highly co-operative event, but the enthalpy change associated with folding and unfolding resembles that of intact lysozyme when their differences in thermal stability are taken into consideration. The significance of these results in terms of the folding process of lysozyme is discussed. By contrast with authentic lysozyme, CM6,127- lysozyme was found to exist in an unfolded state at pH 2 at room temperature. N.m.r. spectroscopy and c.d. were used to characterize this state. Unlike their homologous relative, alpha-lactalbumin, which exists in a partially folded molten globule state under these conditions, only residual non-native-like structure persists in the acid-unfolded state of CM6,127-lysozyme. These results indicate that the difference in folding behaviour of lysozyme and alpha-lactalbumin cannot be accounted for simply by their differences in thermal stability.
1 . The Influence of Proline Residues on Alpha-helical Structure
Woolfson, DN; Williams, DH.
FEBS Letters 277 (1-2) 185-188 (1990)
Proline lacks an amide proton when found within proteins. This precludes hydrogen bonding between it and hydrogen bond acceptors, and thus often restricts the residue to the first four positions of an alpha-helix. Helices with proline after position four have a pronounced kink [(1988) J. Mol. Biol. 203, 601-619]. In these cases, we find that the proline residue almost always occurs on the solvent exposed face of each helix. This positioning facilitates the compensatory hydrogen bonding between solvent and residues P-3 and P- 4 (relative to proline, P), through the formation of the kink. Further, it aids in the packing of long helical structures around globular protein structures.