Available online at www.sciencedirect.
com
ScienceDirect
Synthetic biomolecules
Editorial overview
Derek N Woolfson and Shang-Cheng Hung
Current Opinion in Chemical Biology 2013, 17:925–928
For a complete overview see the Issue
Available online 6th November 2013
1367-5931/$ – see front matter, # 2013 Elsevier Ltd.
All rights reserved.
http://dx.doi.org/10.1016/j.cbpa.2013.10.021
Derek N Woolfson
School of Chemistry, University of Bristol,
Cantock’s Close, Clifton, Bristol BS8 1TS, UK
School of Biochemistry, University of Bristol,
University Walk, Bristol BS8 1TD, UK
e-mail: d.n.woolfson@bristol.ac.uk
Dek Woolfson took his first degree in Chemistry
at the University of Oxford. He then did a Ph.D.
at the University of Cambridge followed by
post-doctoral research at University College
London and the University of California,
Berkeley. After 10 years as Lecturer through to
Professor of Biochemistry at the University of
Sussex, he moved to the University of Bristol in
2005 to take up a joint chair in Chemistry and
Biochemistry. His research has always been at
the interface between chemistry and biology,
applying chemical methods and principles to
understand biological phenomena. Specifically,
his group is interested in the challenge of
rational protein design, how this can be applied
in synthetic biology and biotechnology, and
with a particular emphasis on making
biomaterials for applications in cell biology and
medicine.
Shang-Cheng Hung
Genomics Research Center, Academia
Sinica, 128, Section 2, Academia Road,
Taipei 115, Taiwan
e-mail: schung@gate.sinica.edu.tw
Shang-Cheng Hung obtained his Ph.D. in
Chemistry from the National Tsing Hua
University, Taiwan in 1992. After completing
postdoctoral work at UC Berkeley and The
Scripps Research Institute, he started his
independent research at Academia Sinica in
1998 He is currently a Distinguished Research
Fellow at the Genomics Research Centre,
Academia Sinica. His research interests focus
on carbohydrate chemistry and chemical
biology, particularly those concerning heparan
sulfate and mycobacterial cell envelope
components.
www.sciencedirect.com
This issue of Current Opinion in Chemical Biology covers both polypeptides
(peptides and proteins) and carbohydrates. It brings together chemical
biology, that is, understanding the chemistry and physics of biological
molecules and assemblies; and the design and engineering methods and
principles that are being developed for and applied to these diverse types of
biomolecule. The articles that we gathered highlight the potential for these
molecules in biotechnology and synthetic biology through the fundamental
understanding of basic principles and providing improved methods of design
and manipulation. With this collection of reviews, we trust that readers will
acquire timely ideas and inspirations that ultimately usher new develop-
ments in these burgeoning and exciting fields of study.
Peptide and protein engineering
The peptide and protein engineering section starts with a review that puts
the field in perspective in two respects: first, that one goal should be
functional designs (in this case enzyme-like functions); and second, that
rational polypeptide engineering is non-trivial and far from routine. Feld-
meier and Höcker map out both the recent successes, and remaining
challenges in achieving enzyme-like activities through rational protein
engineering and design. Approaches to this problem are described, which
often include the combination of computational methods to get into pro-
ductive regions of sequence-structure space first, followed by combinatorial
experiments such as directed evolution to refine and improve activities.
These have been used to target protein-catalysed transformations for ester
hydrolysis, retroaldol, Kemp elimination, Diels–Alder, and Morita–Baylis–
Hillman reactions. Despite the growing successes in this embryonic area,
much remains to be learned and addressed. The authors pinpoint some of
these challenges, which include: assessing and predicting conformational
changes in proteins that accompany mutagenesis; the need for improved
methods to engineer substrate binding and orientation within active sites
accurately; and the necessity to consider and incorporate the roles of
‘second-shell residues’ in binding and activity. We look forward to further
progress in this area, which would undoubtedly impact on biotechnology and
synthetic biology if enzyme-like activities can be tailored de novo, or existing
enzyme can be repurposed to order.
Linking the functional and structural aspects of peptide and protein design
and engineering, Peacock’s review reminds us of the important structural
and catalytic roles played by metals in natural proteins, and that we will
really have to capture this in protein engineering and design to make
progress. The emphasis is unapologetically on helical bundles based on
coiled-coil designs. This is because there are good sequence-to-structure
relationships (‘design rules’) to allow the predictive and reliable design and
Current Opinion in Chemical Biology 2013, 17:925–928
926 Synthetic biomolecules
engineering of these structures, and, as a result, more
recent success with these motifs compared with all b-
motifs and mixed a/b-motifs. This focus also allows for
detailed descriptions of some of the designs, which
cover the incorporation of metallo-porphyrins, mono-
nuclear and multi-nuclear sites as well as miscellaneous
metallo-proteins. These involve a wide variety of metals
and ligands, which take up both structural and functional
roles. This article underlines the breadth of possibilities
for this sub-field of protein design and the prospects for
the wider field if we can get aspects of metal binding such
as selectively, control of coordination shells, and reactiv-
ity right.
Next, Jerala and co-workers take us on a whistle-stop tour
of recent progress towards the rational engineering and de
novo design of closed peptide-based and protein-based
assemblies; that is, rather than fibrous, sheet or crystalline
materials. The main concepts for achieving such closed
constructs have been around for a while: namely, combine
symmetry elements from the folded structures of pep-
tides and proteins sensibly, and the resulting constructs
should close spontaneously; for instance, combining
dimeric and trimeric units back-to-back should render
hexagonal units that can be tiled to give sheets, or, with a
slight twist, closed polyhedra. The tour includes protein-
based tetrahedral and octahedral assemblies produced via
computationally directed engineering of natural protein
interfaces; through the de novo design of coiled-coil build-
ing blocks (or toolkits as they are becoming termed under
the influence of synthetic biology) for assembling com-
plex discrete nanoscale objects from the bottom-up; and
onto a single-chain recombinant polypeptide that encode
multiple coiled-coil components to direct the folding of
the first protein tetrahedron. The progress in this area in
the past year alone has been striking, and the future looks
extremely exciting.
Related to this, Sinclair describes recent successful efforts
to use symmetry in folded protein to arrange many
protein structures precisely in space. The area borrows
from crystallography, but here the emphasis is on control
and programming of assembly rather leaving it to the
hapchance of natural physical processes. This is an emer-
ging area largely because of recent advances that have
been made; the idea has been around for a while, and is
probably best attributed to Padilla and Yeates. The con-
cept is simply to use symmetry elements set up by
defined protein oligomers to direct, perpetuate and con-
trol multiple peptide or protein interactions to furnish
large discrete assemblies, fibres and order 2D and 3D
arrays. With these in hand, functional and useful assem-
blies, materials and arrays can be envisaged. As Sinclair
points out, there are many alternate ways that protein
modules can be combined to effect such supramolecular
assembly. The strategies employed include: encoding all
of the information for folding and assembly in a single
Current Opinion in Chemical Biology 2013, 17:925–928
‘poly-peptide/protein’ chain (as reviewed by Jerala and
co-workers); combining small peptide-based building
blocks through non-covalent intermolecular interactions;
and mediating peptide/protein interactions with small-
molecule or metal ligands. Now that considerable pro-
gress is being made in these underpinning aspects of this
new field — that is, in directing and controlling (i.e.,
programming) self-assembly — the question is, what
can supramolecular peptide and protein designers put
their skills and imaginations at work at to create?
Staying with protein–protein interactions (PPIs), Schue-
ler-Furman and co-authors describe how disrupting such
interfaces are being targeted in rational design. These
were once considered ‘undruggable’, but small-molecule
inhibitors of PPIs are now being tackled increasingly
successfully. As part of this effort, Schueler-Furman
and co-authors propose that the concept of ‘hot spots’
in PPIs, originally proposed by Clackson and Wells, can
be embellished leading to the idea of ‘hot segments’.
These are ‘continuous peptide stretches that dominate the PPI’.
As such, they provide footholds for both computation
studies (to estimate binding and to design PPI inhibitors)
and as a guide in subsequent experimental studies. For
instance, these extended regions of PPIs provide a basis
for the rationalisation of existing experimental data, and
for new designs of both small molecule and peptide-based
inhibitors of PPIs. The review also gives some perspect-
ive on the experimental strategies for stabilising peptides
engineered to bind protein surfaces and disrupt PPIs.
The question is, can this spark a new wave in drug
discovery for hitherto intractable targets?
Moving on from discrete and arrayed polypeptide assem-
blies towards fibrous structures, in two separate pieces
Jalan and Hartgerink and Li and Yu describe progress that
has been made towards model collagen-like assemblies
and functional collagen-based materials. First, Jalan and
Hartgerink detail recent advances in understanding and
designing collagen assemblies using model-peptide sys-
tems. They place this in the context of past and current
understanding of collagens as one of the best-known and
oldest-known protein structures, but one that still pre-
sents considerable obstacles in terms of design, pro-
duction and assembly. The review neatly summarises
the sequence, structural and topological challenges in
designing rope-like collagens, which at first sight appears
to be simple, indeed almost trivial protein fold. At
the heart of this new perspective are pairwise salt-bridge
interactions between polypeptide chains, which brace
and specify chain–chain interactions. In turn, these pro-
vide ‘rules for protein design’, which can and have been
used to direct the assembly of homo-typic and hetero-
typic collagen-like model peptides. Continued studies
and advances in this area are likely to result in the
programming of self-assembled collagen-based fibrous
and hydrogel materials with potential applications in
www.sciencedirect.com

3D cell culture and tissue engineering. This takes us
nicely onto the next topic in the section.
Functionalising assembled collagen fibres and materials is
the meat and potatoes of Li and Yu’s review. In addition
to the above of course, collagens are the most abundant
mammalian protein, and they are key components of the
extracellular matrix and connective tissues. As a result,
producing and/or mimicking collagens could lead to un-
derstanding and correcting certain diseases, and to bio-
medical applications in wound healing, cosmetics and
regenerative medicine. The review centres on ‘col-
lagen-mimetic peptides’ (CMPs), which are small pep-
tides that hybridise with partly unfolded or remodelled
collagen materials. CMPs can be used to adorn existing in
vivo collagens with new functions, or to bring function to
bare collagen scaffolds both natural and de novo. These
CMPs bind or otherwise incorporate into the assembled
collagens through mechanisms that are not fully under-
stood. However, this has been achieved using heat-
unfolded CMPs and partly unfolded collagens. As heating
is not ideal for living tissues, alternative methods are
being studied such as accessing unfolded CMPs through
caging and subsequent UV exposure to trigger release of
the active peptides; and accessing native collagens during
normal remodelling in vivo. Achieving selectivity or speci-
ficity of binding to the many endogenous collagens is also
not trivial as is discussed. Bringing this type of work to
fruition could see genuine real-life applications.
In final shift in topic, this time from aqueous to membrane
environments for proteins and protein design, Simms and
Booth talk us through the perils and promise in design
membrane-spanning peptides and proteins. They
describe how this is one of the most demanding areas
for protein engineering and design because of the lack of
high-resolution data on transmembrane proteins, our lack
of understanding of the forces at play in folding and
stabilising their structures, and the difficulties in handling
these molecules. Though difficult and complex targets,
progress is being made, which includes new and
improved forcefields for in silico modelling, folding and
design of membrane proteins; taking water-soluble struc-
tures into the membrane; and repurposing natural mem-
brane proteins for new functions. It is clear that this is an
area for near-future advances and one to watch. Cleverly,
the authors also turn the problem around and discuss the
ways in which water-soluble variants of membrane-span-
ning proteins are being engineered to allow detailed
structure-function studies of these otherwise intransigent
but extremely important class of biomolecule.
Carbohydrate synthesis, design and
applications
Sugars offer dense structural information mainly due to
the multiple chiral centres and linkage potentials of each
residue. Their ubiquity in the cell surface also presents
www.sciencedirect.com
Editorial overview Woolfson and Hung 927
prospects for multivalency — considered a hallmark of
carbohydrate–protein interactions. Wittmann presents
the difficulties and the approaches that have been devel-
oped to elucidate multivalent interactions, focusing on
carbohydrate as the ligand. He describes the possible
binding modes that complicate the study of such systems.
Distances between binding sites have been evaluated
using molecular rulers that, in combination with several
analytical techniques, supplied much needed details sur-
rounding the binding event. In particular, electron para-
magnetic resonance spectroscopy detected the distances
between binding sites of nitroxide-labelled carbohydrate
ligands.
The quest for large and complex synthetic glycans is the
focus of the article by Lowary. While short fragments
derived from a large glycan often suffice in understanding
bioactivity, many cases exist wherein the desired
responses are only produced using large structures. Gen-
erating such materials is far from trivial despite the
various advances in constructing carbohydrates in recent
years. The assembly of complex N-glycans, the glyca-
nated erythropoietin, complex glycolipids, and com-
pounds that could already be considered as
polysaccharides are among the examples provided. Such
endeavours are opportunities in honing the skill and tool
sets in oligosaccharide synthesis that are poised to match
the established protocols for today’s protein assembly.
Human immunodeficiency virus (HIV), the causative
agent of AIDS, heavily relies on its ‘glycan shield’ in
evading the human body’s immune defences. The dis-
coveries of carbohydrate-specific broadly neutralising
antibodies (bnAbs) that are specific for glycans situated
in the outer envelope of HIV glycoproteins and the
slippery road towards the characterisation and synthesis
of the glycan epitopes, ultimately for vaccine design are
summarised by Wang. With the revelation of more than a
dozen bnAbs, the various designs of the carbohydrate
ligands, including their conjugation to carrier proteins and
virus-like particles, are explored. This promising avenue
for vaccine development has yet to bear significant fruit
and thus remains an area of active investigation.
Guo and co-workers cover the current developments in
the synthesis and bioevaluations of glycosylphosphatidy-
linositol (GPI) anchors and GPI-anchored proteins. GPI
anchors are quite common in eukaryotic cell membrane
and have profound effects in the activity of the anchored
protein. Chemical strategies that permit the efficient
assembly of GPI anchors in their various forms have been
reported. Native chemical ligation and the enzyme sor-
tase A are options available in delivering GPI-anchored
peptide/proteins although both techniques introduce
unnatural components to the construct. The authors also
propose various directions that could be taken to further
gain insights on GPI anchorage on the cell surface.
Current Opinion in Chemical Biology 2013, 17:925–928
928 Synthetic biomolecules
Moving on another challenging glycan target, two separ-
ate reviews highlight the synthesis and bioevaluations of
glycosaminoglycans. Griffin and Hsieh-Wilson report on
the synthetic methods on preparing glycosaminoglycan-
derived oligosaccharides and glycomimetic polymers.
The authors focus particularly on notable efforts concern-
ing heparan sulfate/heparin and chondroitin sulfate, the
two most prominent members in this family of polyanionic
polysaccharides. Additionally, Hung and co-authors pre-
sent a more detailed look at the recent tactics applied to
prepare heparin and heparan sulfate oligosaccharides as
well as the knowledge gained from studying their inter-
actions with selected proteins. Many proteins depend on
the fine structures of these sugars to properly function,
which make them attractive targets in biomedical research.
Recent advances in chemical and chemoenzymatic
Current Opinion in Chemical Biology 2013, 17:925–928
syntheses allowed the preparation of a significant sampling
of the enormous structural possibilities available. These
compounds have already assisted in shaping the knowl-
edge on the kinetic, thermodynamic and molecular details
of the interactions with several proteins.
Sialic acids are commonly found at the outermost ends of
conjugated glycans. Fittingly, the final article on this
edition is on the synthesis of sialic acid-containing sac-
charides. Here, Lin and co-workers focus on the
approaches that deal with difficult a-stereoselectivity in
sialylation, which are then applied in the assembly of
sialylated glycans including the oligosialic acids. Prep-
arations of unnatural glycan linkages and the enzyme-
functionalised magnetic nanoparticle-mediated synthesis
are also described.
www.sciencedirect.com