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Biomedical subjects

Derek N Woolfson

Publications and source records attributed to Derek N Woolfson.

At least 19 recordsLinked to original sources
PubMed2006

Kinking the coiled coil--negatively charged residues at the coiled-coil interface.

The coiled coil is one of the most common protein-structure motifs. It is believed to be adopted by 3-5% of all amino acids in proteins. It comprises two or more alpha-helical chains wrapped around one another. The sequences of most coiled coils are characterized by a seven-residue (heptad) repeat, denoted (abcdefg)(n). Residues at the a and d positions define the helical interface (core) and are usually hydrophobic, though about 20% are polar or charged. We show that parallel coiled-coils have a unique pattern of their negatively charged residues at the core positions: aspartic acid is excluded from these positions while glutamic acid is not. In contrast the antiparallel structures are more permissive in their amino acid usage. We show further, and for the first time, that incorporation of Asp but not Glu into the a positions of a parallel coiled coil creates a flexible hinge and that the maximal hinge angle is being directly related to the number of incorporated mutations. These new computational and experimental observations will be of use in improving protein-structure predictions, and as rules to guide rational design of novel coiled-coil motifs and coiled coil-based materials.

Aspartic Acid↗
PubMed2006

Peptide-based fibrous biomaterials: Some things old, new and borrowed.

Bioinspired fibrous materials that span the nano-to-meso scales have potentially broad applications in nanobiotechnology; for instance, as scaffolds in 3D cell culture and tissue engineering, and as templates for the assembly of other polymer and inorganic materials. The field is burgeoning, and this review is necessarily focused. It centres on recent developments in the design of peptide-based fibres and particularly those using the alpha-helix and the collagen triple helix as building blocks for self-assembly. Advances include new designs in both categories, the assembly of more-complex topologies using fibres themselves as building blocks, and the decoration of the assembled materials with functional moieties.

Biocompatible Materials↗
PubMed2006

Synthetic ligands for apo-neocarzinostatin.

Neocarzinostatin (NCS) is a 1:1 complex of an enediyne chromophore (NCSChrom), non-covalently bound to an 11 kDa protein (apoNCS). We are exploring apoNCS as a generic protein system for sequestering small molecules for therapeutic applications. Here, we disclose a new flavone ligand 1 for apoNCS and present a high-resolution NMR structure of this ligand bound to apoNCS. This is the first high-resolution structure of a completely non-cognate ligand bound to the apoNCS protein. This work provides unambiguous evidence that a completely new class of ligand can bind specifically to apoNCS. Furthermore, the mode of binding is different than that of the naphthoate-based ligands, and for such a simple hydrophobic compound, the new ligand surprisingly binds specifically. This work indicates that apo-Neocarzinostatin has multiple selective and distinct binding modes for small-molecule cargo.

Apoproteins↗
PubMed2005

ZiCo: a peptide designed to switch folded state upon binding zinc.

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.

Binding Sites↗
PubMed2005

MaP peptides: programming the self-assembly of peptide-based mesoscopic matrices.

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.

Circular Dichroism↗
PubMed2005

The design of coiled-coil structures and assemblies.

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.

Amino Acid Sequence↗
PubMed2004

Sequence and structural duality: designing peptides to adopt two stable conformations.

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 intramolecularly 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.

Amino Acid Sequence↗
PubMed2004

Design and synthesis of a nitrogen mustard derivative stabilized by apo-neocarzinostatin.

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.

Antineoplastic Agents↗
PubMed2004

Fiber recruiting peptides: noncovalent decoration of an engineered protein scaffold.

Fiber Recruiting (FiRe) peptides are described. These are derivatives of self-assembling fiber (SAF) forming peptides that are conjugated with small molecules (in our case, biotin or a FLAG-tag octapeptide). The FiRe peptides are co-assembled into fibers and used as bait to recruit folded and functional proteins to the fiber surfaces. This was demonstrated using two molecular recognition models: namely, a protein-ligand interaction (biotin-streptavidin) and an antigen-antibody (FLAG octapeptide-anti-FLAG-antibody) interaction. This concept offers an approach to mimicking in natural fibrillar systems, such as collagen or fibrin, that communicate specifically with their environments via incorporated or decorated active peptide and protein components.

Amino Acid Sequence↗
PubMed2004

Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin.

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, I13F, 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 A 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.

Amino Acid Sequence↗
PubMed2004

Biophysical and mutational analysis of the putative bZIP domain of Epstein-Barr virus EBNA 3C.

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-Jkappa 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-Jkappa in vivo by 50%, and perturbation of the charge on the basic region completely abolishes this function of EBNA 3C.

Active Transport, Cell Nucleus↗
PubMed2003

"Belt and braces": a peptide-based linker system of de novo design.

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, while 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 approximately 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.

Amino Acid Sequence↗
PubMed2003

Chemical synthesis and cytotoxicity of dihydroxylated cyclopentenone analogues of neocarzinostatin chromophore.

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.

Antibiotics, Antineoplastic↗
PubMed2003

Extended knobs-into-holes packing in classical and complex coiled-coil assemblies.

This year marks the 50th anniversary of Crick's seminal paper on the packing of alpha-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.

Algorithms↗
PubMed2003

Engineering the morphology of a self-assembling protein fibre.

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 de novo. We previously described a self-assembling system comprising two short complementary segments of straight synthetic polypeptides (termed 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 biomimetic materials for applications in surface and tissue engineering.

Amino Acid Sequence↗
PubMed2002

Solution structure of a novel chromoprotein derived from apo-neocarzinostatin and a synthetic chromophore.

The natural complex Neocarzinostatin comprises a labile chromophore noncovalently bound to an 11.2 kDa protein. We present the first high-resolution structure of a novel complex derived from the recombinant apoprotein 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 location similar to that observed for the chromophore in the natural Neocarzinostatin complex, but with a distinct orientation. These results provide structural evidence that the apoprotein can readily accommodate small druglike entities, other than the natural chromophore within its binding cleft. The clinical use of the natural complex described by others, together with the results reported here, suggests potential applications for small molecule binding by apo-Neocarzinostatin.

Antibiotics, Antineoplastic↗