Table Of ContentMethods in
Molecular Biology 2208
Maxim G. Ryadnov Editor
Polypeptide
Materials
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
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Polypeptide Materials
Methods and Protocols
Edited by
Maxim G. Ryadnov
National Physical Laboratory, Teddington, Middlesex, UK
Editor
MaximG.Ryadnov
NationalPhysicalLaboratory
Teddington,Middlesex,UK
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
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Preface
This volume continues the misson of the book series to provide the research community
with a systematic overview of contemporary research methods in the area of molecular
biology. The volume details the synthesis and assembly of polypeptide materials (proteins
andpeptides),theirprecursors,conjugates,andderivatives.Aparticularemphasisismadeon
themeasurementtoolsandproceduresfor materialcharacterization,bothphysicochemical
and functional. Often overlooked in the literature, such a focus can address the growing
demand for improving reproducibility in biomedical research. It can also help to better
reflect the interdisciplinary nature of molecular biology and the importance of developing
innovativemeasurementmethodstoadvancethearea.Writtenasstep-by-stepinstructions,
protocols in this volume are not limited to the given examples and are applicable to other
materialsofthesameandsimilartypes.Thisisalsomeanttohelpthereadertokeepabreast
with achievements beyond the scope of each topic avoiding both generalization, which
wouldotherwiseomitimportantnuancesinmeasurementresults,e.g.,sourcesofvariation,
andinstantiation,whichwouldnarrowthemdowntospecialistcases.
The volume opens with a protocol for the chemical synthesis of peptide thioesters.
Theseareroutinelyusedasbuildingblocksforcomplexstructuresrangingfromproteinsto
advanced functional materials (Raz and Offer). Traditional approaches toward peptide
thioesters use highly hazardous chemistries, which are increasingly problematic to imple-
mentinthemodern,risk-averselaboratorypractice,whereasalternativeroutescontinueto
suffer from poor reproducibility, giving products of low purity and yields. All of this is
addressed in the first chapter that describes a shortcut methodology to peptide thioesters,
whichcanenablepost-synthesisapplicationssuchasnativechemicalligation.Unlikechemi-
cal methods, which require achieving optimum stoichiometry, native peptide–peptide liga-
tions may proceed to completion at nearly equimolar ratios of ligating fragments. These
reactionsareself-catalyzed,whiletheirefficiencycanbeimprovedusinganauxiliarymeans
suchasconformationallydrivenligationsthatbringligatingendsincloseproximityallowing
the two components to fuse even at low concentrations. An example of these reactions is
given in the following chapter, which offers a straightforward method for peptide–peptide
ligation (Buldun et al.). The method termed SnoopLigase conjugation facilitates the site-
specificfusionofproteinsviatheformationofanisopeptidebondwith>95%efficiency.The
described strategy relies on the use of peptide tags and has already been demonstrated to
enhanceenzymeresilienceandtheefficacyofvaccinesthroughantigenoligomerization.The
protocol details a range of conditions for SnoopLigase reactions together with optimized
purification steps for the ligated products and the quantification of ligation efficiency. The
keyadvantagesofthismethodarehighligtedtoincludethemaximumconversionofreacting
components,lowconcentrations used,andprecisetaglocationwhichprovetobeeffective
eveninside cells. Both thechapters advocate for covalent conjugatesas preferable forms of
peptidematerials.Thisholdstrueformany.Forother technologiesofbiologicalrelevance,
covalent modifications may not be necessary or even prohibitive, while both covalent and
non-covalent macromolecular systems are equally common. Gene therapy is one of such
application areas. Its main purpose is to deliver functional genetic material into the cell.
Genes, be these RNA or DNA, cannot traverse cellular membranes by themselves and
requireacarrier vehicletomediatethetransfer.Avarietyofpeptideswithsimilarproperties
v
vi Preface
have been shown to provide suitable packaging systems for various nucleic acids and
oligonucleotidesalike.
The next two chapters describe protocols for the preparation and characterization of
non-covalent gene packaging systems. Gomez et al. make stress on nanoparticle formula-
tionsresultingfromcell-penetratingpeptidesthatcaneithercondenseorencapsulatesmall
interferenceRNA(siRNA)andDNA.Theprotocolisexemplifiedbyanumberofcommer-
cial and experimental reagents and provides an exhaustive workflow for their preparation
and analysis including peptide synthesis, purification, efficiency and stability of particle
formation, and intracellular uptake. Welch et al. follows upon this chapter with nanoparti-
culate formulations of siRNA enabled by condensation with bespoke disulfide-constrained
cyclic peptides. The peptides act as a switch system that opens up in the reducing environ-
ments of the cell and is degraded by proteases releasing siRNA into the cytoplasm. This
protocol details a step-by-step preparation of the formulation, from synthesis to siRNA
complexation, and ramps up the biological validation of peptide packaging systems of this
type to animal model experiments, demonstrating effective gene knowdown, which con-
firmstherelevanceandapplicabilityofnon-covalentpeptidesystemsinvivo.Thenatureof
forces that drive nanoparticle formation is not specifically addressed in these two chapters.
Nevertheless, for all the systems described, conformational changes are involved in com-
plexation,intracellular uptake,andrelease.Insomecases,thechangesareadirectresultof
bindingtonucleicacids;inothers,itisthefoldingofpeptidesthatmediatesthecomplexa-
tionorpredeterminesencapsulation.
Complementary to this, Chapter 5 reports quantitative examples of conformationally
drivenfunctionsmediatedbyproteinsandpeptides(Shiuetal.).Thisprotocoldescribesthe
protein-triggered opening of a nanoscale box assembled using the principles of DNA
origami.TheconstructionoftheDNAboxisdetailedinlinewiththefunctionalassessment
of the opening process that is monitored by Fo¨rster resonance energy transfer (FRET).
Lactate dehydrogenase from Plasmodium falciparum specifically binds to a DNA aptamer
servingasa“lock”inthebox.Whenthetargetproteinbindstotheaptamer,theboxopens,
whichleadstoachangeinFRETsignal.Alltheprocessessupportingthemethodologyare
mediated by highly specific molecular recognition events between biological materials of
differentchemistries.Exploitingsucheventsfordevelopingandstudyingfunctionalmateri-
alsisthesubjectofthefollowingchapter whichbroadenstheapplicationofconformational
changes to the behavior prediction of self-assembling systems (Marzuoli and Fraternali).
This chapter presents detailed guidelines for molecular dynamic simulations of biological
nanoscalesystemswithaparticularemphasisontheirhierarchicalnature.Thelatterisneatly
matched with force fields used in simulations, which are categorized according to the
accuracy and speed they offer. Each force field is assigned to a specific type of simulation,
thus probing the pros and cons of their prediction power in comparison, and to a specific
hierarchicaltypeofbiologicalassemblyascomplexasproteincages.
Experimentalprotocolsinemulating,predicting,andexploitingtheproteinshellarchi-
tecture are given in the next four chapters. Chapter 7 (Inaba and Matsuura) describes
nanocapsulate assembly of protein shells from β-annulus peptides. These peptides derive
fromnaturallyoccuringvirusessuchastomatobushystuntvirusandrepresentastructurally
conserved topology allowing for symmetric assembly into spherical hollow nanoparticles.
The protocol details all aspects of their production and analysis starting with peptide
synthesis protocols. Conjugations and non-covalent decorations of the assembled particles
withDNA,metalnanoparticles,andpeptidesarealsodiscussed.Bothexterior(surface)and
interior (encapsulation) functionalizations are presented, rendering this design one of the
Preface vii
mostcharacterizeddiscretesystemsreportedtodate.Thenextchapter(Chakrabortietal.)
takesadifferentapproachwithaprotocoldescribingtheuseofproteincagesinconstructing
regulararraysofmetalnanoparticles.Themethodologyusesthesameprinciplesofelectro-
staticinteractions,butinsteadofencapsulatingmetalparticlesincagesordecoratingcages
with a metal corona, this approach employs the interactions to build three-dimensional
(3D)geometricobjects,inwhichproteincagesandmetalparticlesalternateformingsuper-
lattices. Protein cages with biological activities may present an obvious choice for design.
Thisiscoveredinthefollowingprotocol(Tarasovetal.),whichdescribessyntheticanalogs
of a portion of a transmembrane domain of G-protein-coupled receptors (GPCR). The
analogsaredesignedtoassembleintovirus-likeparticlesretaininganintrisicGPCRinhibi-
toryactivity.Themethodologytakesastructure-baseddesignroutetoimplementboththe
functions,biological and self-assembly, in theconstituent monomer of theparticles.Com-
prehensive instructions are provided for the structure, design, synthesis, assembly, and
physicochemical characterization of the particles. The final chapter in this quartet
(De Santis and Ryadnov) continues this direction with a protocol for imaging and 3D
reconstruction of virus-like particles using high-resolution and cryogenic electron micros-
copy. Unlike examples used in previous chapters, these particles are de novo capsids con-
structed without prior structural information. Designed from scratch, the capsids need
ultrastructural elucidation to support and improve the design principles. Therefore, this
protocol provides major steps in the structural analysis of the particles as a continuum of
measurementprocedures,fromassemblyto3Dreconstruction.
Virus-likeparticlesrepresentoneofthemajornanomaterialmorphologiesthatfinduse
invariousapplicationsandshowconsistentprogressinnewdesignsandthedevelopmentof
bespokemethodologiestoadvancetheprecisionandaccuracyofanalysis.Sincemost,ifnot
all, proteinand peptide nanoscale materials rely onself-assembly processes, methodologies
thatareabletomonitortheprocessesinrelationtoresultingmorphologiesareofparticular
interest.Inthisregard,thenextprotocoldesribestheapplicationoftime-resolvedemission
spectroscopy to track the protein assembly exemplified by fibrillogenesis (Alghamdi et al.).
Proteinfilamentsconstituteanother major nanomaterialmorphology,whichcanalsobeof
natural and synthetic origin. The protocol is built around case studies for both types and
extends measurements to post-assembly events such as aggregation, with relevance to
pathological processes including amyloidosis. The method follows changes in fluorescence
as a function of time, which gives a continuous measure of growth kinetics and, when
applicable,aggregation(e.g.,conversionofamyloidfibrilstoplaques).Representativetime-
resolvedspectrawithdatainterpretationarealsodiscussedtodemonstratetheadvantagesof
themethodology.
Thereexistsasubtleborderlinebetweenthedifferentoutcomesofpeptideassemblyand
aggregation. The former assumes a higher level of control to enable the fabrication of
reproducible materials that deliver the desired functions. The rate and type of assembly is
defined at the primary structure level but can also be directed by external stimuli and
fabrication methods. An exemplar method is described in the next chapter (Cross et al.).
This protocol focuses on the fabrication and characterization of nanostructured hydrogel
materials. Typically designed to support 3D cell culture, both in vitro and in vivo, peptide
hydrogels result from the formation of 3D filamentous networks. Filamentous assembly
does not necessarily lead to gelation. Therefore, successful hydrogel designs are often
complemented with effective fabrication methods. The protocol provides an additional
valueofproducinghydrogelsindifferentforms—thinfilmspreparedinsecondsandthicker
gels prepared in minutes. Protein assembly is initiated within 1 h, but can mature to an
viii Preface
equilibrium phase in a few hours. Monitoring nanomaterial formation under assembly
conditions from the outset is important and is of particular interest if it allows to probe
sizechanges.Suchchangesareauniversalindicatornotonlyforthegrowthkineticsbutalso
for the quality of the produced material as they can inform preventive measures against
undesiredprogessions,e.g.,aggregation.
Dynamiclightscatteringisacommontoolforsizemeasurements,withyetlimiteduse
due to the requirements of using large sample volumes and low throughput. These chal-
lengesareaddressedintheprotocolbyKwanetal.,whichdescribesdynamiclightscattering
measurements executed in a fully automated fashion for low-volume samples and in solu-
tion, i.e., in situ. The protocol offers a generic pre-screening method for downstream
structural studies of polypeptide and other biomolecular materials using higher-resolution
approachesincludingX-raycrystallography,electronmicroscopy,small-angleX-rayscatter-
ing,andNMR.Sinceinmostcasesnanoscalepeptidematerialsareproducedunderaqueous
conditions,solutionmeasurementsremainaprerequisitefor theircharacterization.Finally,
assembledproductscanbeusedunderdifferentconditions,whichnecessitatethedevelop-
ment of other specialist methods and protocols. Peptide coatings are increasingly relevant
forapplicationswhereaccuratecontrolover thickness,homogeneity,anddensityisimpera-
tive.X-rayphotoelectronspectroscopyoffersoneofthemostinformativemethodstoprobe
theseproperties.Thistechniqueishighlysensitivetochangesonsurfacesprovidingquanti-
tativeinformationonthechemicalcompositionofmaterialswithin10-nmdepthsfromtheir
surfaces.Theuseofthismethodforflatand3Dsubstrates(e.g.,nanoparticulatematerials)is
detailed in the protocol by Cant et al. Surface analysis approaches are explored for more
complex measurements aiming to characterize different events and under different
conditions.
Atomic force microscopy (AFM) has revolutionalized the area of protein assembly at
interfacesandisrapidlyprogressingtowardlivecellandhigh-speedmeasurementsofvarious
biologically relevant meachanims mediated by peptide materials (Hammond et al.). This
development is characteristic of a timely and complementary response to advances in
material designs and biotechnologies that demand more precise mechanistic investigations
in native and near-native environments. Broader developments concern microscopy cap-
abilities as a whole. One drive here is to enable correlative imaging of the same biological
processes or systems using microscopy modalities that differ by imaging speed, resolution,
andsamplesize.Correlativeimagingisviewedasanultimatesolutiontothecharacterization
of nanoscale materials in cellular media. AFM is one of such modalities. It can cover the
nano-to-micrometer length scale but has limitations in depth resolution and screening
throughput. Other approaches lack the resolution of AFM but can support high-
throughput measurements, ensuring statistically significant analysis of cell populations,
while being able to support single-cell measurements. A highly effective strategy is to
empower optical fluorescence microscopy with microfluidic devices such as one described
in the penultimate protocol of the volume (Cama and Pagliara). Here, a single chip of
thousands of micro-channels can accommodate thousands of individual cells whose
response to peptide treatment can be imaged over a set period of time. This allows kinetic
measurements of cell responses (growth and lysis) as well as concentration-dependent
peptide–cell interactions. Exemplified by antimicrobial effects of peptide materials on bac-
terial phenotypes, the strategy is adaptable to other cell-based measurements including
processesthatseeknewinsightsattheintracellularlevel.However,revealingorconfirming
the details at this level demands much higher resolution, whereas the number of available
modalities is limited. Encouragingly enough, ultramicrotomy analysis of peptide-treated
Preface ix
cells using electron microscopy proves to provide access to critical information at highest
resolution possible today. Described in the last chapter by Rey et al., this methodology
provides a step-by-step instruction for sample preparation and processing, imaging and
analysisofbacterialandmammaliancellsfollowingpeptidetreatment.Furthermore,exam-
ples of intracellular infections and their targeting by bespoke peptide materials is also
discussed, thus culminating this volume with arguably the most challenging and complex
applicationmeasurementofpeptidematerialsatthenanoscale.
Teddington,Middlesex,UK MaximG.Ryadnov
