Peptide Polymer Conjugates 2013

PC64CH28-Xu ARI 28 February 2013 19:6

Peptide-Polymer Conjugates:From Fundamental Scienceto ApplicationJessica Y. Shu,1 Brian Panganiban,1 and Ting Xu1−3

1Department of Materials Science and Engineering and 2Department of Chemistry,University of California, Berkeley, California 94720-1760;email: [email protected] Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,California 94720

Annu. Rev. Phys. Chem. 2013. 64:631–57

First published online as a Review in Advance onJanuary 16, 2013

The Annual Review of Physical Chemistry is online atphyschem.annualreviews.org

This article’s doi:10.1146/annurev-physchem-040412-110108

Copyright c© 2013 by Annual Reviews.All rights reserved

Keywords

hybrid biomaterial, self-assembly, polymer conformation, protein-polymerinteraction

Abstract

Peptide/protein-polymer conjugates make up a new class of soft mattercomprising natural and synthetic building blocks. They have the poten-tial to combine the advantages of proteins and synthetic polymers (i.e., theprecise chemical structure and diverse functionalities of biomolecules andthe stability and processability of synthetic polymers) to generate hybridmaterials with properties yet to be realized with either component alone.Here we briefly discuss recent developments in the design, fundamental un-derstanding, and self-assembly of various peptide-polymer conjugates, aswell as emerging biological and nonbiological applications that range fromnanomedicine, to separation, and beyond.

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Peptides: shortoligomers comprisingmonomers known asamino acids

Proteins: linearpolymers of aminoacids that are muchlonger than peptidesand describe complete,biologicalmacromolecules with astable conformation

Peptide-polymerconjugate: hybridbiomacromoleculecomprising syntheticpolymers covalentlyattached to peptides

Self-assembly:process in whichdisordered systemsform organizedstructures as aconsequence ofspecific, localinteractions among thecomponents, withoutexternal direction

Secondary structure:local, specific,geometrical shape of apeptide; typicalsecondary structuresare the α-helix and theβ-sheet

Tertiary structure:three-dimensionalstructure of a foldedprotein formed fromthe packing ofsecondary structuralelements

Amphiphile:chemical compoundcomprisinghydrophilic andhydrophobicproperties

1. INTRODUCTION

The materials community has been striving for decades to generate biomimetic materials toaccess properties seen in nature. However, there has been limited success in achieving hierarchicalstructural control, long-term enzymatic activity, selective molecular transport, and modulatedresponsiveness to small perturbations, to say nothing of control over energy flow within aclosed system and information flow over time and space. Peptides and proteins, nature’s ownbuilding blocks, are polymers programmed with monomeric resolution that enable most of theproperties listed above. With recent developments in polymer chemistry, a basic understanding ofpolymer phase behavior at the single-chain level, site-specific protein modification, and syntheticbiology, new opportunities have emerged. Rather than mimicking nature, it may be feasible togenerate materials using nature’s building blocks (i.e., proteins) if protein structure and func-tionalities can be maintained under nonbiological environments required for synthetic materialsprocessing.

Peptide/protein-polymer conjugates are a promising new class of soft materials, as theadvantages of each component can be complementary. Peptides and proteins self-assemble intothe native structures encoded by their primary sequence to support a diverse and complex array offunctions. They provide hierarchical self-assembly over multiple length scales down to the molec-ular level, chemical functionality, selectivity and specificity, and dynamic responses to externalstimuli, all properties of interest to the materials community. However, the physical limitationsof biomolecules, such as their sensitivity to temperature, pH, organic solvents, and degradation,inhibit their practical application. The attachment of suitably selected synthetic polymers couldpotentially mitigate these limitations by mediating interactions between the proteins and thelocal medium. By combining the hierarchical structure and chemical functionality of the peptidewith the stability and processability of the polymer, peptide/protein-polymer conjugates maypotentially lead to materials of both high complexity and high modularity (1–13). For example,peptide-polymer conjugates can form a system capable of responsive hierarchical self-organizationover at least three length scales. On the smallest length scale, peptide sequences direct folding intoregular secondary structures. Depending on the sequence, some peptides form tertiary structures.Finally, microphase separation between the peptide/protein and synthetic polymer offers organi-zation at a larger length scale. Such hybrid materials may contain novel structures and functionsto meet demands in a wide range of biological and nonbiological applications. However, for thesebuilding blocks to reach their full potential, it is important that the natural structures and functionsof the proteins be maintained upon conjugation of synthetic polymers and during materialsprocessing. A fundamental understanding of various energetic contributions governing the phasebehavior of hybrid conjugates under different environments is essential. This is the foundationon which materials scientists can begin to tailor and enhance the properties of hybrid conjugatesfor specific applications, as well as to enrich our knowledge to develop new materials based onproteins.

Studies in the field of hybrid biomaterials over the past decade or two have ranged from fun-damental science (understanding and controlling the interactions between the two components),to self-assembly of these building blocks (for the generation of hierarchical, biofunctional nano-structures), to biomedical and nonbiological applications (Figure 1). Below we discuss highlightsin these four areas to outline the breadth of the field. Moreover, as peptide/protein-polymer con-jugates are the focus of this review, we do not discuss research in the fields of peptides aloneand of lipopeptides, also known as peptide amphiphiles, although they are clearly closely relatedand have made significant contributions to the field of bioinspired materials (14, 15). Rather, thisoverview focuses on the progress made when attaching synthetic polymers to biomolecules and

632 Shu · Panganiban · Xu

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Fundamentalscience

Peptide/polymer conformation

Stimuli-responsiveness

Interactions between components

Self-assembly

Solution

Bulk

Thin film

Polar/nonpolar interface

Biomedicalapplications

Proteintherapeutics Drug delivery

Tissueengineering

Immunology

Directedbiomineralization

Biosensing

Nonbiologicalapplications

Energy harvesting

Catalysis

Separation

Sequestration

Effect of polymer conjugation

Peptide-polymer

conjugates

Peptide-polymer

conjugates

Figure 1Diagram displaying the four areas of research concerning peptide-polymer conjugates, namely fundamentalscience, self-assembly, and biomedical and nonbiological applications. Each circle contains select examplesof each field, and the arrows signify the interrelation and interdependence between them.

the interesting questions and applications that arise upon merging the biological and syntheticworlds.

This review begins with a concise overview of the various synthetic routes available for gen-erating peptide-polymer conjugates. Much emphasis is placed on understanding the energeticlandscape of these new hybrid biomaterials, and studies invested toward this end are highlighted.Such information is important for the implementation of these materials in hierarchical nanostruc-tures with eventual applications. The self-assembly behavior, along with functional materials basedon these building blocks, is outlined to provide a map of the potential structures and applications.Finally, we conclude with perspectives and a future outlook of the field.

2. SYNTHESIS OF PEPTIDE/PROTEIN-POLYMER CONJUGATES

The variety of peptide/protein-polymer conjugates is immense, as there is flexibility in the lengthand complexity of the protein sequence, the chemical nature, the length and architecture of thepolymer, and the overall architecture of the conjugate (Figure 2). In addition to the vast library ofpeptides and proteins found in nature, de novo design provides the opportunity to generate novelsequences with functions not observed naturally. Synthetic polymer chemists have also generateda wide array of monomers, providing vast modularity of the synthetic block with tailored physical,chemical, mechanical, optical, and electronic properties. Synthetic routes to generate peptide-polymer conjugates are outlined in Figure 2 and are briefly discussed here. The reader is directedto more detailed, comprehensive synthetic reviews elsewhere (16–18).

www.annualreviews.org • Peptide-Polymer Conjugates 633

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Incr

easi

ng le

ngth

/com

plex

ity

S

Various architectures

Polypeptide

Designedpolypeptide

Protein

CH3CH3CH3

CH3H3C

H3C

NO

FOO

O VRRFPWWWPFLRR

n CO2

H3C

CH3

CH3

R1

Y1

RR

HN

OO

O

O

Onn

H+

O

O

OHNHO

n

NH

NH

X

Increasing hydrophobicity

O

O

Linker

R1

Y1

O

ONH

H

R2

Y2

O

ANH

X

Linker

R1

Y1

O

O

ONXH

NH

HN

OO

Y2

R2

H2N

H2O

HSS–R

Linker

Peptide B

OH2N

Peptide B

Peptide A

O

SPeptide A

X = temporary amine protecting groupY = permanent side-chain protecting groupA = carboxy activating group

O

O

Peptide B

SH

Peptide A

+

n

On

CH3H3C

OHNn

H3C

CH3

OO

n n n

O

On

O

NBr

HCH3

O

NBr

HCH3

O

S

CH3S

ATRP

Pn–X + Cu1+/L Pn–PmX–Cu2+/L + P•n

kact

kp

Monomer

kt

kdeact

RAFTS S–Pm kadd

k–add

k–add

kadd

P•n + +

kp

Monomer

P •m

kp

Monomer

S–PmPn–S SPn–S

NH

NH

CH3CH3CH3

CH3

CH3

CH3

H3C

H3C

H3C

NO

O

O OO

FOO

O VRRFPWWWPFLRR

O

NH

NH

n

Methods of peptide synthesisRing-opening polymerization

Nucleophileor base

Deprotection of a-aminopreprotecting group

Coupling of nextamino acid

Intramolecular S-N acyl shift

Peptide macroinitiator

NMP macroinitiator

“Graft to”

Peptide-reactivepolymerLys: –COOH, –NHSCys: –maleimideEtc.

O

NBr

HCH3

Peptide ATRP initiatorO

NS

HCH3S

Peptide RAFT agent

Polymer-peptide conjugate

“Graft from”

Peptide/protein Polymer

Solid-phase peptide synthesis

Native chemical ligation

Protein expression

Conjugation routes

Cleavage of peptideoff support

Controlled radicalpolymerization

Figure 2Overview of the various routes available for the synthesis of peptide-polymer conjugates of tailored compositions, architectures, andsize. Figure reprinted with permission from Reference 16. Copyright 2009 Elsevier.


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2.1. Peptide/Protein Synthesis

In general, the method of peptide synthesis depends on the length and complexity of the peptidesequence. For simple sequences based on a single or a few amino acid species, investigators typicallyuse ring-opening polymerization of N-carboxyanhydrides of protected α-amino acids initiatedby primary amino-end functionalized synthetic polymer macroinitiators (5). As the complexityof the sequence increases, solid-phase peptide synthesis is routinely used to prepare sequence-specific peptides below 50 amino acids in length (19). With recent optimization of solid-phasepeptide synthesis, peptides of well over 60 amino acids have been achieved for certain sequences.Alternatively, native chemical ligation can be used to couple two preformed peptides to formmodified proteins of moderate size, up to 200 amino acids in length (20). Finally, for the synthesisof large proteins, recombinant DNA methods are usually employed (21). However, it remains asignificant challenge to express amphiphilic and hydrophobic peptides.

2.2. Polymer Synthesis and Conjugation

For the synthesis of polymers, many types of polymerization chemistries exist for the generationof polymers of varied composition, controlled molecular weight, and low polydispersity. Thecoupling of polymers to peptides to create conjugates is separated broadly into two strategies,grafting to and grafting from, each of which can occur in solution or in the solid phase (22).An end-functionalized polymer can be grafted to a peptide via complementary chemically activehandles, or a peptide can be used as a macroinitiator from which the synthetic polymer can bepolymerized via one of several different chemistries, such as nitroxide-mediated polymerization(23, 24), atom transfer radical polymerization (23–26), or reversible addition-fragmentation chaintransfer polymerization (22, 26).

2.3. Challenges: Synthesis and Characterization

Although there have been numerous advances in protein synthesis, controlled radical polymeriza-tion, and site-specific conjugation, which together have contributed to the generation of conjugatesof various sizes, compositions, and architectures, a few synthetic hurdles still remain to preparehybrid conjugate materials. Achieving complex conjugates of multiple components and chemi-cal modifications is challenging, as they are more difficult to purify, which limits the quantityand yield of the target conjugate and increases the cost and time of production. In particular,standard purification and characterization methods, such as reversed-phase high-pressure liquidchromatography and matrix-assisted laser desorption-ionization time of flight, respectively, arenot trivial for complex amphiphilic conjugates.

3. ENERGETIC CONSIDERATIONS OF PEPTIDE/PROTEIN-POLYMERCONJUGATES

3.1. Energetic Balance Between Protein-Polymer Interactionsand Polymer Chain Conformation

The phase behavior of peptide-polymer conjugates is dominated by a variety of noncovalent inter-actions, such as van der Waals interactions, hydrogen bonds, hydrophobic interactions, electro-static interactions, π-π interactions, and the entropy associated with polymer chain configurations.A thermodynamically stable conformation arises from the minimization of the overall free energy


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C CN+

H

R

H

H

H O–

O

0

Entropy of deforming an ideal chain

Entropic spring constant

N: degree of polymerization

b: Kuhn length

k—–– + S(N,0)32

R→2

Nb2

Uij

–ε

σ r

r

y

x

z

R→

dR→y

dR→x

dR→z

a b

e

c

d S(N,R

→) = – –

3kT/(Nb2)

Figure 3Energetic considerations of peptide-polymer conjugates. (a) Chemical structure of amino acids, whose sidegroup, R, defines the interactions between each residue pair. (b) Protein sequences, as depicted by the chainof circles of various colors, determine the location and composition of each R group and their relativepositions (i.e., interaction-distance relationship). (c) Energetically, it is the combination of multiple pairs ofthese rather weak interactions that stabilizes individual protein structures and ensures proper proteinfolding. These secondary interactions, such as van der Waals interactions, electrostatic interactions, andhydrophobic interactions, have strong distance dependences and dissipate quickly as inter–amino aciddistances increase, as shown in the graph that plots energy versus distance. (d ) Schematic representation ofthe various intermolecular interactions involved with peptide-polymer conjugates dissolved in solvent media,namely intrapeptide, peptide-polymer, peptide-solvent, and polymer-solvent interactions. Proteins,polymers, and solvent molecules are represented by the orange, green, and white circles, respectively.(e) Polymer conjugation affects the local polymer chain conformation, which reflects a delicate balancebetween the polymer-residue-solvent interactions and the entropic polymer chain conformation.

Primary structure:sequence of aminoacids in a peptide orprotein, typically readfrom the N to Cterminus

resulting from all the intra- and intermolecular interactions, and engineering materials with de-sired properties requires achieving a delicate balance of all these energetic contributions. Fornatural proteins, the side group, R, of each residue (Figure 3a) defines the interactions betweeneach residue pair that are generally in the range of a few kilocalories per mole. Energetically, itis the combination of multiple pairs of these rather weak interactions that stabilizes individualprotein structures and ensures proper protein folding. These secondary interactions have strongdistance dependence and dissipate quickly as inter–amino acid distance increases (Figure 3c). Theprotein sequence, or primary structure (Figure 3b), essentially determines the location and com-position of each R group and their relative positions (i.e., interaction-distance relationship). Weakinteractions can be readily strengthened or broken, leading to stimuli responsiveness, and localconditions (such as solvent, pH, and ionic strength) can modulate the conformational propertiesof the conjugate.

To build hybrid biomolecular materials, investigators position synthetic polymers nearproteins. The monomer-residue distance is well in the range such that amino acid–monomer


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PEG: poly(ethyleneglycol)

Coiled coil: commontertiary motif, aleft-handed supercoilof multipleright-handed α-helices

interactions are equally important as interresidue interactions. The protein cannot distinguishwhether the monomer is part of the protein. A similar situation holds for protein-solvent andpolymer-solvent interactions (Figure 3d ). Upon attaching polymers, the effects of protein-polymer interactions on protein structure depend on the chemical nature of the locally surroundingpolymer and the spatial distribution of monomers near the protein. In turn, protein-polymerinteractions also affect the local polymer chain conformation. Experimental and theoreticalstudies have shown that the local polymer chain conformation reflects a delicate balance betweenthe polymer-residue-solvent interactions and the entropic polymer chain conformation. Withina certain range of molecular weights, attached polymers can arrange locally.

3.2. Fundamental Studies

For protein/peptide-polymer conjugates to reach their full potential as useful materials, the struc-ture and function of the peptide/protein should be maintained upon polymer conjugation. Towardthis end, it is important to understand the effect of conjugating polymers on peptide/protein struc-ture, dynamics, and function and the effect of the biomolecule on polymer chain configurationand its materials properties. The effects are dependent on a variety of factors, such as the natureof the polymer, the chemical heterogeneity of the peptide, the site of polymer conjugation, andthe solvent media. Success in achieving desirable, functional assemblies relies on understandingthe interactions between each building block and delicately balancing and manipulating them toachieve targeted assemblies without interfering with designed structures and functionalities. Suchstudies, as outlined in Figure 4, have contributed to our understanding of the energetic landscapeof peptide-polymer conjugates.

3.2.1. Polymer effect and molecular weight dependence. The most studied peptide-polymerconjugates are of the linear diblock architecture, in which a synthetic polymer is conjugated to apeptide terminus. For example, when poly(ethylene glycol) (PEG) is linked to the end of coiledcoils (Figure 4a) (6, 27–29), its presence enhances the stability of the peptide structure againsttemperature and pH changes. However, depending on the length of the peptide sequence and thehelical propensity, conjugating the PEG chain to the N terminus reduces the peptide helicity andlowers the degree of association of the bundle (29). This effect is more obvious as the peptide’soligomeric state and the polymer molecular weight increase and the peptide length decreases(28, 30). It is speculated that the attachment of PEG to the N termini of the coiled coil restrictsthe volume available to the polymer chains, leading to molecular crowding and thus favoringlower aggregation states, as well as the unwinding of the ends of the helices. The linear analog isexpected to undergo greater loss in peptide structure as the molecular weight of PEG increases.Furthermore, the influence of the length of PEG on a de novo designed peptide-PEG conjugate,which exhibits a calcium-ion-sensitive transition from random coil to α-helix, was investigated:the longer the PEG block, the greater the concentration of Ca2+ necessary to stimulate the coil-to-helix transition. A careful balance of stabilizing and destabilizing effects in the peptide is requiredto realize rapid responsiveness, and PEG’s stabilization of secondary structures is polymer lengthdependent (31).

3.2.2. Architecture dependence. The architecture of conjugates is another important parameterwith which to address fundamental questions. Whereas most conjugates are of the linear diblockvariety, examples of peptides bearing polymeric side chains attached at the middle of the peptide(Figure 4c) are less common (12). Side conjugation of PEG to the exterior of coiled coilsled to stabilization of peptide secondary and tertiary structures and preservation of built-in


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Fundamental studies

En

d c

on

jug

ate

S

ide

co

nju

gat

e

Hydrophilic polymer Hydrophobic polymer

Polymer conformation Protein conformation& activity

or

Conjugate conformation

0 ns 5 ns 20 ns

a

c

e f g

b

dEffect of polymer

conjugation

Interaction betweencomponents

ΔC, ΔT ΔC, ΔT

Figure 4Examples of fundamental studies of peptide-polymer conjugates, including (a) hydrophilic end conjugates, (b) hydrophobic endconjugates, (c) hydrophilic side conjugates, and (d ) hydrophobic side conjugates, that address such questions as the effect of polymerconjugation and the interaction between components. Other studies include the characterization of (e) polymer chain conformation,( f ) protein conformation and activity, and ( g) the conformation of the conjugate as a whole. Panel a reprinted with permission fromReference 29. Copyright 2003 American Chemical Society. Panel b reprinted with permission from Reference 37. Copyright 2010American Chemical Society. Panel c reproduced from Reference 138 by permission of The Royal Society of Chemistry. Panel dreprinted with permission from Reference 139. Copyright 2011 Wiley. Panel e reprinted with permission from Reference 47.Copyright 2011 American Chemical Society. Panel f reprinted with permission from Reference 39. Copyright 2002 Wiley. Panel greprinted with permission from Reference 49. Copyright 2011 American Chemical Society.

functionality within the interior of the helix bundle. In this case, increasing PEG’s length(up to 5,000 Da) increases the helical content of the peptide and enhances its stability againsttemperature changes. Detailed structural information on the side conjugates was extracted usingsmall-angle X-ray scattering (SAXS) and a newly developed model in which each peptide-PEGconjugate was represented as a Gaussian chain attached to a cylinder, which was further arrangedinto a bundle-like configuration of multiple cylinders (32). The side conjugates were found toretain helix bundle structure, with the polymers slightly compressed in comparison with theconformation of free polymers in solution. In contrast, when conjugated to the end of coiled coils,the radius of gyration of the PEG chains was found to be similar to the unperturbed dimensionof free chains in solution (R. Lund, J. Shu & T. Xu, unpublished results). Molecular dynamic


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PMMA: poly(methylmethacrylate)

simulations of side conjugates confirmed helix stabilization upon PEG conjugation and showedthat the oxygens of PEG interacted favorably with the cationic lysine side chains on the exteriorof the helix bundle, which may reasonably account for the compact configuration of the PEGchain revealed by SAXS (33). Such studies show that the conformation of the polymer whenconjugated to the peptide is dependent on a balance of the enthalpic interactions between thepolymer and the peptide and the entropy of the polymer chain.

Similar to the architecture of side conjugates, alternating multiblocks of PEG can be conju-gated to peptides through the f position on the heptads by hetero- and homodimeric associationof coiled coils. The coiled coils not only retained their capacity to form hetero-oligomers, butalso gained the ability to form homo-oligomeric micelles upon PEGylation (34). Even less inves-tigated are peptides containing more than one polymer side chain. A recent study presented thesynthesis of peptide-polymer conjugates containing well-defined linear peptide backbones with adefined number of polymeric side chains. Depending on the composition, number, and length ofthe polymer side chains, the conjugates aggregated to form different topologies (35). In addition,responsive diblock and triblock copolymers of protein-polymer conjugates have been synthesizedby grafting polyethyleneglycol methacrylate (PEGMA) and PEGMA-pPEGMA from trypsin,respectively. They exhibited different solution behavior with temperature, dependent on the ar-rangement of the grafted polymer blocks. Enzyme activity also varied with hybrid architecture,demonstrating the ability to generate conjugates of varied structures, architecture, and solutionbehavior (36). The great flexibility afforded by the conjugate architecture provides a means totailor the interactions between the components. This also expands the library of available buildingblocks suitable for a wide array of applications.

3.2.3. Amphiphilicity dependence. The incorporation of hydrophobic moieties to create am-phiphilic conjugates can lead to self-assembly of hierarchically structured, biomolecular materials.Systematic understanding of how the peptide structure and functionality are affected upon incor-porating hydrophobic moeities is required to direct assemblies in solution, in solid state, and atinterfaces. However, depending on the peptide sequence and native structure, studies have shownthat the hydrophobic moieties affect peptide structures differently. The attachment of a polystyrenechain to the N termini of a heme-binding coiled coil showed that the presence of hydrophobicpolystyrene partially unfolded the peptide secondary structure and consequently compromisedthe binding pocket within the core of the bundle owing to hydrophobic polymer-peptide inter-actions at the peptide/polymer interface (Figure 4b) (37). Such effects depend strongly on theperiodicity of hydrophobic residues and on interhelix interactions. When covalently linked topolystyrene, Candida antarctica, horseradish peroxidase, and myoglobin showed decreased enzymeactivity (Figure 4f ). This was attributed to hydrophobic polystyrene-induced destabilization ofthe active conformation of each enzyme, disturbed cofactor binding in the apo-protein, or reducedaccess of the substrate to the active site of the protein (38–40). In addition, extensive studies haveshown that proteins adsorbed onto hydrophobic surfaces denature and lose their enzymatic activi-ties owing to hydrophobic interactions (41–43). A finding observed across many peptide-polymerconjugate systems is that the interfacial zone between the peptide and the synthetic polymer con-sists of amino acid residues that do not form regular secondary structures (44). Therefore, it maybe beneficial to vary the architecture of the conjugate or the site of polymer conjugation to retainpeptide structure and designed functionality.

The effects of the polymers on the peptide structure and function have been systematicallyinvestigated as a function of varying degrees of hydrophobicity of the conjugated polymer,showing a direct correlation between the polymer hydrophobicity and the peptide structurein aqueous solution. Researchers studied hydrophobic side conjugates by varying the degree


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PNIPAAM: poly(N-isopropylacrylamide)

of hydrophobicity of the side-conjugated polymer, from polystyrene, PMMA [poly(methylmethacrylate)], to PNIPAAM [poly(N-isopropylacrylamide)]. The presence of the hydrophobicpolymer unfolded the peptide helices and induced an α-helix to β-sheet conformational transition(Figure 4d), leading to deleterious effects on the binding pocket of the heme-binding helix bundle.These effects decreased as the polymer became less hydrophobic. The peptide helical structureswere retained by adding organic solvents that solubilized the hydrophobic polymers. Theseresults reiterate that the protein structure in a peptide-polymer conjugate reflects competitionbetween the peptide-polymer interactions and the polymer-solvent interactions and is sensitiveto the local environment. It is feasible to dissolve amphiphilic peptide-polymer conjugates inorganic solvents to enhance their solution processability while maintaining protein structure.

3.3. Challenges

Future progress toward gaining a fundamental understanding of hybrid biomaterials will relyon a combination of the types of studies described above, along with more challenging detailedstructural characterization and computer simulations that have generally been lacking to date.We consider two areas of knowledge that are key to further our basic understanding of the phasebehavior of protein-polymer conjugates. One is the polymer chain conformation upon conjugationto the protein. This requires detailed structural characterization of protein-polymer conjugates.The other is how the local protein-polymer-solvent interactions affect the local arrangements ofprotein structures and polymer segments. In addition to various techniques used to probe proteinstructures, molecular dynamics simulations or coarse-grained simulations, for example, are neededto provide key insights to design polymers and conjugates.

PEG’s interaction with proteins has been investigated to determine its conformation whenconjugated to proteins (Figure 4e). The most commonly presumed structure is a shroud model,in which the attached PEG chain wraps around the protein to create a shielding effect (45–47).Studies that indicate reduced immunogenicity of proteins upon PEGylation assume a shroudmodel. A shroud model was also presumed in the analysis of a size-exclusion chromatographystudy (45) and a SAXS study of PEG-antibody conjugates, in which PEG is thought to effectivelycover part or all of the antibody (46). These observations support the view that PEG interactswith proteins to form an exterior shell. In an alternate model, however, PEG does not interactsignificantly with the protein. Rather, the conjugate forms a dumbbell-like architecture, in whichtwo noninteracting entities are covalently linked (47, 48). In recent small-angle scattering studiesthat investigated the configuration of PEG conjugated to lysozyme, human growth hormone, orhuman galectin-2, a dumbbell configuration was observed, with Rg of conjugated PEG similar orgreater to that when free in solution (47, 48). Furthermore, a model that combines the dumbbelland shroud views was observed in SAXS studies of PEG-hemoglobin (48) and coiled-coil-PEGside conjugates. These conflicting observations may result from the properties of a PEG-proteinconjugate depending on a variety of factors, such as the site of conjugation, the molecular weightof PEG, the number of PEG chains per protein, and the chemical heterogeneity of the surfaceof the protein. Furthermore, extracting such information from an experimental point of view ischallenging, even with scattering techniques, and clear conclusions may be difficult to make.

Computer simulations are vital in garnering information unobtainable from experiment aloneand for interpretation of experimental results. Molecular dynamic simulations (Figure 4g) mayprovide a means to determine the local arrangements of segments from proteins and polymers thatcannot be readily extracted experimentally (49, 50). Simulations, in conjunction with systematicexperimental studies as described above, provide a means to address fundamental issues that willcontribute to the rational design of these hybrid materials.


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Enzyme b

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c Temperature

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T < LCST T > LCST

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Figure 5Responsiveness of peptide polymer conjugates to various stimuli, including changes in (a) pH, (b) enzymatic activity, (c) ion/cofactorbinding, and (d ) temperature. Panel a reprinted with permission from Reference 146. Copyright 2005 American Chemical Society.Panel b reprinted with permission from Reference 52. Copyright 2010 American Chemical Society. Panel c provided by Professor HansBorner. Panel d reprinted with permission from Reference 147. Copyright 2008 American Chemical Society.

4. TOWARD FUNCTIONAL HYBRID BIOMATERIALS: SELF-ASSEMBLY

Peptide/protein-polymer conjugates can take advantage of existing processing techniques andstrategies to direct polymeric nanostructures, while achieving structural control at the atomiclevel that has been elusive with synthetic polymers alone. Various functions, both natural andnonnatural, can be incorporated into the peptide/protein block through sequence design andmodification, and the great monomer selection available for the synthetic block provides theability to mediate the interactions between the peptide and its environment and to control theself-assembled structure in a predetermined way. Furthermore, the peptide’s responsiveness toexternal stimuli, such as pH, temperature (34), enzymes (51, 52), and ion/cofactor binding (31),can be exploited to construct smart materials that undergo a change in size or structure whendesired (Figure 5).


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Polypeptides:peptides of simplesequences typicallycomprising one or afew amino acid species

4.1. Solution

Engineering amphiphilicity into peptide-polymer conjugates enables them to self-assemble intofunctional, biomolecular nanostructures, such as spherical micelles, cylindrical micelles, and vesi-cles, when dissolved in a selective solvent for one block (3, 8, 9, 44, 53–56). A majority of studies havefocused on conjugates comprising hydrophobic synthetic polymers and water-soluble polypeptidesegments that fold into α-helices (44, 53, 57–59). These form a multitude of structures with tun-able morphologies (60–62) and stimuli responsiveness (58, 63–65). In addition, β-sheet-formingconjugates form fibrils directed by peptide organization, with a peptide core and a polymer corona(66), that can reach micrometers in length and display a superhelical fine structure with a left-handed helical twist (67, 68). With increased peptide complexity, a heterodimer coiled coil was usedto noncovalently link PEG and polystyrene blocks, and the resultant amphiphilic PEG-peptide-polystyrene triblock copolymer assembled into thermoresponsive micelles that transformed froma rodlike to spherical structure upon heating (69). In another report, a thermoresponsive tri-block comprising poly(diethylene glycol methyl ether methacrylate) conjugated to the ends of atriple-helix-forming collagen-like peptides displayed a transition from spherical aggregates thatincreased in size with increasing temperature up to 65◦C to fibrils at 75◦C (70). Giant amphiphiles,compounds comprising an entire enzyme or protein as the head group and a hydrophobic poly-meric tail attached to a preselected, well-defined location, are also able to form typical structures insolution (38–40, 71–81). In addition, triblock hybrid copolymer analogs have been synthesized andwere found to form rare structures, such as Y-junctions, toroids, octopus structures, dumbbells,and other aggregates, along with the expected structures (82, 83).

4.2. Polar/Nonpolar Interfaces

Amphiphilic conjugates are also able to assemble at polar/nonpolar interfaces. Langmuir mono-layers of tripeptide-polystyrene diblocks and peptide-polystyrene-peptide amphiphiles were in-vestigated at the air-water interface (84). The architectural variations had a great impact on theself-assembly of the amphiphiles, especially for the transition from the gas phase to the liquidphase, because steric interactions between polymer chains are dominant. However, in a con-densed phase, the packing of the various architectures was comparable (84). A β-sheet-poly(n-butyl acrylate) conjugate spread at the air-water interface showed a β-sheet network that consistsof antiparallel β-sheets oriented parallel to the interface. Stable monolayers with low compress-ibilities formed when β-sheet assembly occurred in monolayers of close-packed conjugates. TheLangmuir-Schaefer transfer of the monolayer seeded with stearic acid resulted in a monolayer con-taining ordered domains with widths in agreement with the end-to-end distance of the conjugate(85). Furthermore, monolayers of an amphiphilic polymer, poly(allylamine), containing poly(L-alanine) hydrophobic graft chains demonstrated strong pH-dependent assembly. In acidic condi-tions, the protonated allylamine moieties dissolved in the aqueous phase, while the hydrophobicgrafts acted as anchors to keep the monolayer at the interface. In alkaline conditions, the polymerwas located entirely at the interface, forming stable and closely packed uniform monolayers owingto dehydration of the alkylamine moieties, with the graft chains forming β-sheet domains (86).α-Helix-forming polypeptide-PEG conjugates spread at the air-water interface were also foundto form stable monolayers, with the polypeptide forming α-helices that were oriented slightlynormal to the interface at high surface pressures. Langmuir-Blodgett monolayers deposited onsolid substrates confirmed that the α-helix of the peptide block was oriented slightly normal tothe surface, although the presence of PEG tilted the rod at the interface, preventing an entirelyupright orientation (87).


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BCP: blockcopolymer

4.3. Bulk

Typical linear block copolymer (BCP)-type homopolypeptide-polymer conjugates, in which thepeptide forms simple secondary structures, self-assemble into morphologies similar to those seenwith fully synthetic BCPs (5, 10, 58, 69). Most α-helical polypeptide-polymer conjugates tendto form hexagonal-in-zigzag lamellar morphologies, with the peptide helices either interdigi-tated or folded and packed hexagonally (53, 58, 59, 88–94). Hexagon-in-hexagonal morphologieshave also been observed (58). β-sheet peptide-PEG conjugates have been found to form lamellarstructures with lyotropic liquid crystal nematic and hexagonal columnar phases and PEG crys-tallization (95, 96). Similar to the case in solution, the β-sheet-forming peptide has a significantimpact on the solid-state structure of the resulting conjugate. Longer PEG spacers lowered thetendency to form antiparallel β-sheets and resulted in microphase separation (97). With largerbiomolecules, protein-PNIPAAM conjugates formed highly disordered lamellae or hexagonallyperforated lamellae. Solvent annealing resulted in a transition toward lamellar nanostructures withproteins packed in a bilayer and improvements in long-range order (98).

4.4. Thin Film

Similar to that observed in bulk, α-helical polypeptide-polymer conjugates also form hexagonal-in-lamellar morphologies in thin films. A recent study presented peptide-polymer conjugates con-taining well-defined linear peptide backbones with a defined number of polystyrene or PNIPAAMside chains. Spin-coated peptide-polystyrene conjugates formed honeycomb structures for smallpolymer chains and coral-like morphologies for longer side chains, whereas peptide-PNIPAAMconjugates aggregated to ring-shaped topologies (35). A thin film of a triblock containing a π-conjugated polymer flanked by two polypeptide blocks retained electroactive and photoactiveproperties of the polymer. The presence of the polypeptide blocks did not impede charge injection,transport, and photophysical processes essential for electroluminescence. Different nanostructureassemblies were observed depending on the copolymer composition and the secondary structureof the polypeptide block (99).

In addition, peptide/protein-polymer conjugates can be coassembled with synthetic BCPs tocreate composites that exhibit hierarchical structural complexity and chemical functionality local-ized to specific microdomains of the BCP thin film. Ferritin-PEG nanoparticles were incorporatedin a poly(2-vinylpyridine)-b-poly(ethylene glycol) thin film. The resulting composite containedferritin-PEG nanoparticles confined to PEG microdomains, showing that direction of proteinlocalization within BCP films is possible. Recently, the simultaneous coassembly of PS-b-PEOwith heme-binding coiled-coil-PEG side conjugate and myoglobin-PEG conjugates was reportedto form hierarchically structured films using organic solvent-based solution processing (Figure 6)(100). Protein structure was retained, heme-binding by helix bundles was reconstituted, and theretention of myoglobin peroxidase activity was observed in thin films. The hierarchical organiza-tion of these multiple functional elements, through a combination of protein folding, biomolecularrecognition, and BCP microphase separation, represents a new approach to fabricate functionalbiomolecular materials using peptides and enzymatically active proteins. This work demonstratesthe feasibility of synchronizing multiple self-assembly processes to achieve hierarchically struc-tured, protein-containing soft materials with molecular-level control.

Conjugates have also been adsorbed onto surfaces. BCPs containing a polypeptide segmentwith alkyl side chains hierarchically self-assemble on graphite through epitaxial adsorption ofthe peptide segment. Morphologies were concentration dependent, ranging from island-likeaggregates, monolayers, and monolayers with larger nanorods to monolayers with ring-shaped


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20 nm

17 nm

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Self-assembly

Solution a

dc

Pb-b-PGA

Air

WaterInwater

Solvent

Copolymer

Lowconcentration

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HOPG

Nanorod MonolayerRing-shapedaggregate

Solid state b

Top view

D = 20 Å

D = 43 Å

d = 5 Å

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d = 16 Åx

z

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aggregates as the concentration increased. These diverse morphologies were a result of hierar-chical assembly induced from peptide α-helix folding, epitaxial adsorption, rod-coil contrast, andamphiphilicity (101).

The various nanostructures formed by self-assembly provide tremendous promise for biomedi-cal and nonbiological applications. These studies, in conjunction with fundamental studies, providedesign principles on which to develop tailored nanostructures with the desired properties to suitspecific applications.

5. TOWARD FUNCTIONAL HYBRID BIOMATERIALS:EMERGING APPLICATIONS

Peptide/protein-polymer conjugates hold great promise for a variety of biomedical applications,including those depicted in Figure 7. Their hierarchical structure, biocompatibility, and stimuliresponsiveness make them amenable to such applications, as they can easily interface with thebiological and synthetic worlds.

5.1. Drug/Gene Delivery

An immense amount of work has been done to generate micelles based on polypeptide-PEGconjugates for small-molecule anticancer drug and gene delivery. Polypeptide-PEG micelles aregenerally tens of nanometers in size with various intermolecular interactions in the core-formingpolypeptide block to regulate micelle stability for prolonged circulation and controlled drug re-lease (102–109). Polypeptides such as poly(aspartate), poly(glutamate), and poly(lysine) have beenutilized. Common anticancer drugs can be incorporated into the core of the micelle through hy-drophobic interactions or covalent attachment to polypeptide side chains. Micelles show prolongedblood circulation in comparison to free drugs by avoiding glomerular filtration and reticuloen-dothelial system uptake and effectively accumulating in the tumor via the enhanced permeationand retention effect. Micelles typically demonstrate higher efficacy with fewer side effects thanfree drugs as well. For the delivery of nucleic acids, one usually employs a polyion complexationbetween anionically charged DNA or RNA and a BCP having a hydrophilic segment and a cationicsegment, such as poly(lysine). The polyplex micelles exhibit efficient gene introduction in culturedcells and also show gene expression in vivo.

Stimuli-responsive micelles have also been generated to release cargo at target sites whendesired based on environmental cues. For example, differences in the concentration of reductiveagents between the extracellular and intracellular environment and pH reduction in endosomescan be used as triggers for drug release to achieve smart nanocarriers. In addition, endosomalescape and cytosolic delivery are especially important for effective delivery. Investigators used thefusogenic activity of a pH-sensitive micelle, PEG-poly(histidine), in endosomes to facilitate the

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 6Examples of the self-assembly of peptide-polymer conjugates (a) in solution, (b) in the solid state, (c) at the air/water interface, and(d ) in thin films. Panel a reprinted with permission from Reference 140, copyright 2003 Springer, and reprinted with permission fromReference 60, copyright 2005 American Chemical Society. Panel b reprinted with permission from Reference 97, copyright 2010American Chemical Society; reprinted with permission from Reference 91, copyright 2002 Elsevier; and reprinted with permissionfrom Reference 59, copyright 2000 American Chemical Society. Panel c reproduced from Reference 84 by permission of The RoyalSociety of Chemistry and reprinted with permission from Reference 85, copyright 2008 American Chemical Society. Panel dreproduced from Reference 100 by permission of The Royal Society of Chemistry and reprinted with permission from Reference 99,copyright 2004 American Chemical Society, and from Reference 101, copyright 2007 American Chemical Society.


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HPMA:N-(2-hydroxypropyl)methacrylamide

cytosolic delivery of cargo. Furthermore, specific ligands, such as antibodies, transferrin, folate,sugars, and peptides, can be appended to the surface of the micelle to achieve active targeting tocancer cells for increased tumor accumulation, increased cellular uptake, and increased transfectionefficiency in vitro.

Despite these many advances, it remains a significant challenge to generate organic nanocarrierswith a long circulation half-life, effective tumor penetration, and efficient clearance of metabolites.A new family of amphiphiles based on coiled-coil 3-helix bundle forming peptide-PEG conjugateshas recently been developed. The amphiphiles self-assemble into monodisperse micellar nanopar-ticles, 15 nm in diameter (110). By incorporating unique protein tertiary structures in the headgroup of an amphiphile, investigators can generate organic nanoparticles with tunable stability,ligand clustering, and controlled disassembly to meet current demands in nanomedicine. Cargocan also be attached to a polymer backbone via coiled-coil linkers formed by the heterodimer-ization of two complementary peptide sequences that are linked to the polymer carrier and thecargo, respectively (28, 111). Not only are the coiled-coil linkers useful for binding and releasingcargo, but they may also play an active role in enhancing and directing intracellular transport andtrafficking (111, 112).

Rather than serving solely a structural function in drug-delivery systems, peptides can also servea therapeutic function. For example, a micellar nanocontainer delivery and release system was gen-erated based on β-sheet-PEG conjugates, in which the core is peptidic and the corona is PEG.Enzymatic degradation using chymotrypsin near the conjugation site led to micelle disassemblyand release of peptides in unaggregated forms. This demonstrates the potential in the targeteddelivery of peptides for therapeutics applications (52). Conjugates containing coiled coils as a thera-peutic molecular switch have also been developed. PEG-FosW was able to form a stable coiled-coilheterodimer with the target c-Jun sequence of the oncogenic AP-1 transcription factor (112).

5.2. Protein Therapeutics

The most commonly used polymer in hybrid biomaterials is PEG. PEGylation has proven to bean effective strategy to enhance the kinetic stabilization of proteins, to obtain catalytic activityat very high temperatures, and to improve the stability, pharmacokinetics, and biodistributionof therapeutic proteins (2, 113, 114). FDA-approved PEGylated proteins include Adagen, On-caspar, Krystexxa, PEGASYS, and PEG-INTRON (115). The unique chemical properties ofPEG, such as its high solubility, amphiphilicity, and inertness, are key to its effectiveness (2,116–118). Most studies have focused on the improved pharmacological performance of PEGy-lated proteins compared with their unmodified counterparts and their differences in bioactivity.Variables such as the molecular weight of PEG, the number of PEG chains per protein, the ar-chitecture of the polymer (branched or linear), and its site of conjugation can affect the in vivohalf-life and the activity of proteins. Significant work has also been done with protein-HPMA [N-(2-hydroxypropyl)methacrylamide] conjugates. HPMA is a hydrophilic, biocompatible polymer

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 7Examples of biomedical applications of peptide-polymer conjugates, including (a) protein therapeutics, (b) drug and (c) gene delivery,and (d ) tissue engineering. Panel b reproduced from Reference 108 by permission of The Royal Society and reprinted with permissionfrom Reference 110, copyright 2012 American Chemical Society. Panel c reproduced from Reference 144 by permission of the NaturePublishing Group. Panel d reprinted with permission from Reference 141, copyright 2009 Wiley, and reprinted with permission fromReference 145, copyright 2011 Elsevier.


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whose α-carbon substitution and N-substituted amide bond ensure the hydrolytic stability of theside chains. Results are similar to those garnered with PEG (119–122).

Thermoreversible protein-PNIPAAM conjugates have also been well studied. Similar toPEG-ylated and HPMA analogs, PNIPAAM conjugates exhibit retention of activity, even higherthan the native enzyme, and improved thermal stability (123, 124). However, they can also be sep-arated from solution by thermal precipitation. Streptavidin-PNIPAAM conjugates can reversiblyblock biotin association as the polymer’s conformation changes at its lower critical solution tem-perature, or be engineered to repeatedly bind biotin at room temperature and release boundbiotin at the lower critical solution temperature as the temperature is cycled (125, 126). Poly-mer conjugation to proteins has made a large impact on protein therapeutics and in generatingstimuli-responsive biomolecules with tailored activities and solubilities.

5.3. Nonbiological Applications

Aside from the typical biomedical applications usually associated with peptide-polymer conju-gates, these materials are also capable of impacting nonbiological applications, ranging from gasseparation to optoelectronics and catalysis (Figure 8). For instance, peptide-polymer conjugatescan be used to generate porous thin films containing subnanometer channels oriented normalto the surface, which exhibit unique transport and separation properties and can serve as selec-tive membranes for separation and protective coatings. A new approach has been developed inwhich the growth of cyclic peptide nanotubes can be directed in a structural framework set bythe self-assembly of BCPs (127). By conjugating polymers to cyclic peptides, the subunit of anorganic nanotube can be selectively solubilized in one copolymer microdomain. The conjugatedpolymers also mediate interactions between nanotubes and the local medium and guide the growthof nanotubes in a confined geometry. This led to subnanometer porous membranes containinghigh-density arrays of through channels. This new strategy takes full advantage of the nanoscopicassembly of BCPs and the reversibility of organic nanotube growth and circumvents impedimentsassociated with aligning and organizing high-aspect-ratio nano-objects normal to the surface.

Oligopeptide-oligothiphene hybrids, which form nanostructured fibrillar aggregates, arepromising for optoelectronic applications because of the self-assembly and stimuli responsivenessprovided by the peptide, combined with the semiconducting properties of the thiophene block(50, 128, 129). They represent a novel class of biomimetic materials, rendering well-orderedoptoelectronic segments by the self-assembly of biological moieties, to eventually generatefunction by the structuring of materials. The photophysical changes observed upon assemblyhighlight the strong electronic communication within the amyloid-like aggregates, which aremediated by π-stacking among molecular components, a critical component necessary forcharge transport or exciton delocalization in nanostructures of semiconductive π-systems (130).Fibrillar superstructures are promising if combined with existing top-down technologies, suchas plotting or printing processes, because this should allow for a rational design of macroscopicmaterials with anisotropic optoelectronic properties and hierarchically ordered fine structures(68). Furthermore, researchers have achieved the macroscopic alignment of peptides bearingπ-conjugated functionality via a solution method to process self-assembling peptide amphiphilesinto highly aligned macrostructures (hydrogel “noodle” formation technique) (131), whichresults in materials displaying unique photophysical and anisotropic electrical responses. Theresulting hydrogel noodles resulted in alignment of the π-electron oligomers and led to globaldirectionality among the internal π-stacked chromophores. The anisotropic electrical propertiesof the organic semiconductors buried within the noodles led to an increase of an order ofmagnitude in hole mobilities along the length of the macrostructure (132). In addition, a


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Nonbiological applications

Gas separation a

d

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c

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Directed mineralization/nanoparticle growth

Catalysis

20 nm

Tether polymerto 8CP

Blend 8CP-polymerconjugates with BCP

Grow 8CP nanotubeswithin BCPmicrodomains

3 μm 100 nm

Peptideβ-sheet core

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Block copolymer (BCP)

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Figure 8Examples of nonbiological applications of peptide-polymer conjugates, including (a) gas separation, (b) optoelectronics, (c) catalysis, and(d ) directed mineralization and nanoparticle growth. Panel a reprinted with permission from Reference 127. Copyright 2011 AmericanChemical Society. Panel b reprinted with permission from Reference 129, copyright 2008 Wiley, and reprinted with permission fromReference 130, copyright 2008 American Chemical Society. Panel c reprinted with permission from Reference 142. Panel d (directedmineralization) reprinted with permission from Reference 143, copyright 2008 Wiley, and (nanoparticle growth) reproduced fromReference 134 by permission of The Royal Society of Chemistry.


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polypeptide-oligothiophene conjugate, which formed organogels with the peptide adoptingan α-helical conformation, was used to fabricate organic photovoltaic and organic field-effecttransistor devices by deposition of the PCBM-blended active layer at concentrations shown toinduce self-assembly of the polymer. These devices showed significantly greater hole mobility,short circuit current, and efficiency compared with control compounds. This work establishesthe potential of these hybrid materials to increase device performance (133).

Investigators achieved the controlled formation of silver nanoparticle arrays with high particledensity and short interparticle spacing by using supramolecular nanotapes of oligopeptide-PEGconjugates as templates to direct the nucleation and growth of silver nanoparticles. The optical andelectronic properties of nanoparticles make them desirable for applications such as light trappingin solar cells, which require control of their organization. The hierarchical organization providedby the oligopeptides allows for organization of nanoparticles at smaller length scales than thosecurrently achieved with synthetic BCPs alone (134).

Finally, vesicles made from protein-polymer conjugates can be used to generate bionanoreac-tors for catalysis. Vesicles comprising HRP-PS or HRP-PMMA encapsulating GOx were shownto undergo cascade reactions, in which GOx oxidized glucose to form H2O2, which was subse-quently used by HRP to oxidize ABTS (135). A nanocontainer was also formed from atom transferradical polymerization–mediated in situ preparation of BSA-PS giant amphiphiles. A one-pot hi-erarchical incorporation of enzymes was achieved without interfering with the protein integrityof the overall aggregate. The nanocontainers are permeable and can be used as nanoreactors thatdisplay catalytic activity (136). Although biomedical applications of peptide-polymer conjugatesare more thoroughly studied in comparison to nonbiological applications, the large library ofproteins found in nature provides immense opportunities for the practical utilization of variousfunctions, and their hierarchical organization allows for the generation of nanostructures with anorder on length scales smaller than that possible with synthetic polymer alone.

6. PERSPECTIVES AND OUTLOOK

Protein/peptide-polymer conjugates clearly hold great promise as an interesting class of materialswith a wide range of applications. Because these conjugates are hybrid materials that combine thebiological and synthetic worlds, research in this area often lies at the interface between many dis-ciplines. Such work ranges from fundamental materials science and polymer physics to chemicalbiology, structural characterization in real and reciprocal spaces, and cell biology. From a materialsscience point of view, a fundamental understanding of these hybrids materials is necessary, alongwith control over their self-assembly into well-defined nanostructures, if these building blocks areto reach their full potential in biological and nonbiological applications. Because many nonco-valent interactions of similar energy scales underlie the behavior of peptide-polymer conjugates,a delicate balance of the various energetic contributions must be achieved to reach targeted as-semblies. Detailed structural characterization is also necessary to fully deduce the structure of thepeptide and the polymer chain conformation. With fundamental structural and behavioral prop-erties understood, the knowledge gained can then be used to rationally design peptide-polymerconjugates with desired properties. Many parameters can be tailored to achieve a target, includingthe peptide sequence and length, the chemical nature of the polymer, the length of the polymer,the solvent (depending on the application), and the site of conjugation (or the architecture of theconjugate). When applying these materials to applications, it is important to engineer well-definedmaterials whose structure and behavior are well characterized. This will aid in understanding andtailoring the behavior of these materials in various environments. Toward this end, scatteringis a powerful tool used to deduce the structure of peptide-polymer conjugates. Research that


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combines detailed structural characterization with application performance is important for fu-ture progress. The complexity and diversity of protein/peptide-polymer conjugates are obvious.Computation and simulation are in critical need to provide valuable guidance toward moleculardesign and to develop basic understanding in their phase behavior, not only in their static 3Dstructures, but also in terms of dynamics and kinetic pathway. In this way, hybrid biomaterials,such as protein/peptide-polymer conjugates, may begin to reach their potential.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We acknowledge support from the Office of Science, Office of Basic Energy Sciences, of the USDepartment of Energy under contract DE-AC02-05CH11231 ( J.Y.S. and T.X.) and the Office ofthe Army of the US Department of Defense under contract W91NF-09-1-0374 (B.P. and T.X.).We would like to acknowledge all the authors who have sent us original figures for republicationin this article.

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PC64-FrontMatter ARI 28 February 2013 12:13

Annual Review ofPhysical Chemistry

Volume 64, 2013Contents

The Hydrogen Games and Other Adventures in ChemistryRichard N. Zare � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Once upon Anion: A Tale of PhotodetachmentW. Carl Lineberger � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Small-Angle X-Ray Scattering on Biological Macromoleculesand Nanocomposites in SolutionClement E. Blanchet and Dmitri I. Svergun � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �37

Fluctuations and Relaxation Dynamics of Liquid Water Revealedby Linear and Nonlinear SpectroscopyTakuma Yagasaki and Shinji Saito � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �55

Biomolecular Imaging with Coherent NonlinearVibrational MicroscopyChao-Yu Chung, John Boik, and Eric O. Potma � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �77

Multidimensional Attosecond Resonant X-Ray Spectroscopyof Molecules: Lessons from the Optical RegimeShaul Mukamel, Daniel Healion, Yu Zhang, and Jason D. Biggs � � � � � � � � � � � � � � � � � � � � � � 101

Phase-Sensitive Sum-Frequency SpectroscopyY.R. Shen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 129

Molecular Recognition and Ligand AssociationRiccardo Baron and J. Andrew McCammon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 151

Heterogeneity in Single-Molecule Observables in the Studyof Supercooled LiquidsLaura J. Kaufman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 177

Biofuels CombustionCharles K. Westbrook � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 201

Charge Transport at the Metal-Organic InterfaceShaowei Chen, Zhenhuan Zhao, and Hong Liu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 221

Ultrafast Photochemistry in LiquidsArnulf Rosspeintner, Bernhard Lang, and Eric Vauthey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

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Cosolvent Effects on Protein StabilityDeepak R. Canchi and Angel E. Garcıa � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 273

Discovering Mountain Passes via Torchlight: Methods for theDefinition of Reaction Coordinates and Pathways in ComplexMacromolecular ReactionsMary A. Rohrdanz, Wenwei Zheng, and Cecilia Clementi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 295

Water Interfaces, Solvation, and SpectroscopyPhillip L. Geissler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 317

Simulation and Theory of Ions at Atmospherically Relevant AqueousLiquid-Air InterfacesDouglas J. Tobias, Abraham C. Stern, Marcel D. Baer, Yan Levin,

and Christopher J. Mundy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 339

Recent Advances in Singlet FissionMillicent B. Smith and Josef Michl � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 361

Ring-Polymer Molecular Dynamics: Quantum Effects in ChemicalDynamics from Classical Trajectories in an Extended Phase SpaceScott Habershon, David E. Manolopoulos, Thomas E. Markland,

and Thomas F. Miller III � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

Molecular Imaging Using X-Ray Free-Electron LasersAnton Barty, Jochen Kupper, and Henry N. Chapman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 415

Shedding New Light on Retinal Protein PhotochemistryAmir Wand, Itay Gdor, Jingyi Zhu, Mordechai Sheves, and Sanford Ruhman � � � � � � � � 437

Single-Molecule Fluorescence Imaging in Living CellsTie Xia, Nan Li, and Xiaohong Fang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 459

Chemical Aspects of the Extractive Methods of Ambient IonizationMass SpectrometryAbraham K. Badu-Tawiah, Livia S. Eberlin, Zheng Ouyang,

and R. Graham Cooks � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 481

Dynamic Nuclear Polarization Methods in Solids and Solutions toExplore Membrane Proteins and Membrane SystemsChi-Yuan Cheng and Songi Han � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 507

Hydrated Interfacial Ions and ElectronsBernd Abel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 533

Accurate First Principles Model Potentials forIntermolecular InteractionsMark S. Gordon, Quentin A. Smith, Peng Xu,

and Lyudmila V. Slipchenko � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 553

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Structure and Dynamics of Interfacial Water Studied byHeterodyne-Detected Vibrational Sum-Frequency GenerationSatoshi Nihonyanagi, Jahur A. Mondal, Shoichi Yamaguchi,

and Tahei Tahara � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 579

Molecular Switches and Motors on SurfacesBala Krishna Pathem, Shelley A. Claridge, Yue Bing Zheng,

and Paul S. Weiss � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 605

Peptide-Polymer Conjugates: From Fundamental Scienceto ApplicationJessica Y. Shu, Brian Panganiban, and Ting Xu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 631

Indexes

Cumulative Index of Contributing Authors, Volumes 60–64 � � � � � � � � � � � � � � � � � � � � � � � � � � � 659

Cumulative Index of Article Titles, Volumes 60–64 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 662

Errata

An online log of corrections to Annual Review of Physical Chemistry articles may befound at http://physchem.annualreviews.org/errata.shtml

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Documents

Peptide Polymer Conjugates 2013