Peptide Polymer Conjugates 2013

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Peptide-Polymer Conjugates: From Fundamental Science to Application

Jessica Y. Shu,1 Brian Panganiban,1 and Ting Xu1 3
1
Annu. Rev. Phys. Chem. 2013.64:631-657. Downloaded from www.annualreviews.org by University of Western Ontario on 08/02/13. For personal use only.
Department of Materials Science and Engineering and 2 Department of Chemistry, University of California, Berkeley, California 94720-1760; email: tingxu@berkeley.edu
3 Material Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
Annu. Rev. Phys. Chem. 2013. 64:63157 First published online as a Review in Advance on January 16, 2013 The Annual Review of Physical Chemistry is online at physchem.annualreviews.org This articles doi: 10.1146/annurev-physchem-040412-110108 Copyright c 2013 by Annual Reviews. All rights reserved
Keywords
hybrid biomaterial, self-assembly, polymer conformation, protein-polymer interaction
Abstract
Peptide/protein-polymer conjugates make up a new class of soft matter comprising natural and synthetic building blocks. They have the potential to combine the advantages of proteins and synthetic polymers (i.e., the precise chemical structure and diverse functionalities of biomolecules and the stability and processability of synthetic polymers) to generate hybrid materials with properties yet to be realized with either component alone. Here we briey discuss recent developments in the design, fundamental understanding, and self-assembly of various peptide-polymer conjugates, as well as emerging biological and nonbiological applications that range from nanomedicine, to separation, and beyond.
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1. INTRODUCTION
Peptides: short oligomers comprising monomers known as amino acids Proteins: linear polymers of amino acids that are much longer than peptides and describe complete, biological macromolecules with a stable conformation Peptide-polymer conjugate: hybrid biomacromolecule comprising synthetic polymers covalently attached to peptides Self-assembly: process in which disordered systems form organized structures as a consequence of specic, local interactions among the components, without external direction Secondary structure: local, specic, geometrical shape of a peptide; typical secondary structures are the -helix and the -sheet Tertiary structure: three-dimensional structure of a folded protein formed from the packing of secondary structural elements Amphiphile: chemical compound comprising hydrophilic and hydrophobic properties
The materials community has been striving for decades to generate biomimetic materials to access properties seen in nature. However, there has been limited success in achieving hierarchical structural control, long-term enzymatic activity, selective molecular transport, and modulated responsiveness to small perturbations, to say nothing of control over energy ow within a closed system and information ow over time and space. Peptides and proteins, natures own building blocks, are polymers programmed with monomeric resolution that enable most of the properties listed above. With recent developments in polymer chemistry, a basic understanding of polymer phase behavior at the single-chain level, site-specic protein modication, and synthetic biology, new opportunities have emerged. Rather than mimicking nature, it may be feasible to generate materials using natures building blocks (i.e., proteins) if protein structure and functionalities can be maintained under nonbiological environments required for synthetic materials processing. Peptide/protein-polymer conjugates are a promising new class of soft materials, as the advantages of each component can be complementary. Peptides and proteins self-assemble into the native structures encoded by their primary sequence to support a diverse and complex array of functions. They provide hierarchical self-assembly over multiple length scales down to the molecular level, chemical functionality, selectivity and specicity, and dynamic responses to external stimuli, all properties of interest to the materials community. However, the physical limitations of biomolecules, such as their sensitivity to temperature, pH, organic solvents, and degradation, inhibit their practical application. The attachment of suitably selected synthetic polymers could potentially mitigate these limitations by mediating interactions between the proteins and the local medium. By combining the hierarchical structure and chemical functionality of the peptide with the stability and processability of the polymer, peptide/protein-polymer conjugates may potentially lead to materials of both high complexity and high modularity (113). For example, peptide-polymer conjugates can form a system capable of responsive hierarchical self-organization over at least three length scales. On the smallest length scale, peptide sequences direct folding into regular secondary structures. Depending on the sequence, some peptides form tertiary structures. Finally, microphase separation between the peptide/protein and synthetic polymer offers organization at a larger length scale. Such hybrid materials may contain novel structures and functions to meet demands in a wide range of biological and nonbiological applications. However, for these building blocks to reach their full potential, it is important that the natural structures and functions of the proteins be maintained upon conjugation of synthetic polymers and during materials processing. A fundamental understanding of various energetic contributions governing the phase behavior of hybrid conjugates under different environments is essential. This is the foundation on which materials scientists can begin to tailor and enhance the properties of hybrid conjugates for specic applications, as well as to enrich our knowledge to develop new materials based on proteins. Studies in the eld of hybrid biomaterials over the past decade or two have ranged from fundamental science (understanding and controlling the interactions between the two components), to self-assembly of these building blocks (for the generation of hierarchical, biofunctional nanostructures), to biomedical and nonbiological applications (Figure 1). Below we discuss highlights in these four areas to outline the breadth of the eld. Moreover, as peptide/protein-polymer conjugates are the focus of this review, we do not discuss research in the elds of peptides alone and of lipopeptides, also known as peptide amphiphiles, although they are clearly closely related and have made signicant contributions to the eld of bioinspired materials (14, 15). Rather, this overview focuses on the progress made when attaching synthetic polymers to biomolecules and
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Self-assembly
Solution Polar/nonpolar interface Thin film
Biomedical applications
Drug delivery Tissue engineering Protein therapeutics Biosensing
Bulk
Nonbiological applications Peptidepolymer conjugates

Energy harvesting Sequestration Catalysis
Directed biomineralization Immunology
Fundamental science
Effect of polymer conjugation Peptide/polymer conformation Stimuli-responsiveness Interactions between components
Separation
Figure 1 Diagram displaying the four areas of research concerning peptide-polymer conjugates, namely fundamental science, self-assembly, and biomedical and nonbiological applications. Each circle contains select examples of each eld, and the arrows signify the interrelation and interdependence between them.
the interesting questions and applications that arise upon merging the biological and synthetic worlds. This review begins with a concise overview of the various synthetic routes available for generating peptide-polymer conjugates. Much emphasis is placed on understanding the energetic landscape 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 nanostructures with eventual applications. The self-assembly behavior, along with functional materials based on 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 eld.
2. SYNTHESIS OF PEPTIDE/PROTEIN-POLYMER CONJUGATES

The variety of peptide/protein-polymer conjugates is immense, as there is exibility in the length and complexity of the protein sequence, the chemical nature, the length and architecture of the polymer, and the overall architecture of the conjugate (Figure 2). In addition to the vast library of peptides and proteins found in nature, de novo design provides the opportunity to generate novel sequences with functions not observed naturally. Synthetic polymer chemists have also generated a wide array of monomers, providing vast modularity of the synthetic block with tailored physical, chemical, mechanical, optical, and electronic properties. Synthetic routes to generate peptidepolymer conjugates are outlined in Figure 2 and are briey discussed here. The reader is directed to more detailed, comprehensive synthetic reviews elsewhere (1618).
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Various architectures
Peptide/protein Polymer
Methods of peptide synthesis

Ring-opening polymerization
R n HN O O O Nucleophile or base
Conjugation routes
Graft to
O n HO H3C HN CH3 n CH3 n O CH3 n n O
R H N H n O + n CO2
Solid-phase peptide synthesis

X H O N R1 Y1 H O N R1 Y1 O Linker Deprotection of a-amino preprotecting group O Linker H O N R2 Y2
Peptide-reactive polymer
Lys: COOH, NHS Cys: maleimide Etc.
O O n O
HN H3 C
Graft from
H3 C n O O CH3 S S Cleavage of peptide off support Controlled radical polymerization CH3 H N O Peptide RAFT agent S S O CH3 Increasing hydrophobicity S n
Polypeptide H
Peptide macroinitiator
A Br CH3 H N O Peptide ATRP initiator CH3 H N O
Increasing length/complexity
Coupling of next amino acid Designed polypeptide X N H Y2 R2 H O N
Protein
O Linker O R1 Y1 X = temporary amine protecting group Y = permanent side-chain protecting group A = carboxy activating group
Br
Native chemical ligation

O Peptide A SR O H2N HS H2O O Peptide B RAFT P n + S S Pm kadd k add Peptide B ATRP PnX + Cu1+/L kact kdeact
Polymer-peptide conjugate X Cu2+/L + P n kp Monomer PnS S Pm kadd k add PnS S + kt
Pn Pm
H2N O Peptide A S
Intramolecular S-N acyl shift H O N Peptide A Peptide B O SH
P m
kp Monomer H3 C CH3 CH3 CH3 N O CH3
kp Monomer
Protein expression
+
NMP macroinitiator
O O VRRFPWWWPFLRR N H O
H3 C O N H
O O
O VRRFPWWWPFLRR N H
CH3 CH3 CH3 N O CH3 H3 C n O O CH3 O CH3 F N H3 C H H3C
Figure 2 Overview of the various routes available for the synthesis of peptide-polymer conjugates of tailored compositions, architectures, and size. 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 peptide sequence. For simple sequences based on a single or a few amino acid species, investigators typically use ring-opening polymerization of N-carboxyanhydrides of protected -amino acids initiated by primary amino-end functionalized synthetic polymer macroinitiators (5). As the complexity of the sequence increases, solid-phase peptide synthesis is routinely used to prepare sequencespecic peptides below 50 amino acids in length (19). With recent optimization of solid-phase peptide 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 form modied proteins of moderate size, up to 200 amino acids in length (20). Finally, for the synthesis of large proteins, recombinant DNA methods are usually employed (21). However, it remains a signicant 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 generation of polymers of varied composition, controlled molecular weight, and low polydispersity. The coupling 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 active handles, or a peptide can be used as a macroinitiator from which the synthetic polymer can be polymerized via one of several different chemistries, such as nitroxide-mediated polymerization (23, 24), atom transfer radical polymerization (2326), or reversible addition-fragmentation chain transfer polymerization (22, 26).
2.3. Challenges: Synthesis and Characterization

Although there have been numerous advances in protein synthesis, controlled radical polymerization, and site-specic conjugation, which together have contributed to the generation of conjugates of various sizes, compositions, and architectures, a few synthetic hurdles still remain to prepare hybrid conjugate materials. Achieving complex conjugates of multiple components and chemical modications is challenging, as they are more difcult to purify, which limits the quantity and yield of the target conjugate and increases the cost and time of production. In particular, standard purication and characterization methods, such as reversed-phase high-pressure liquid chromatography and matrix-assisted laser desorption-ionization time of ight, respectively, are not trivial for complex amphiphilic conjugates.
3. ENERGETIC CONSIDERATIONS OF PEPTIDE/PROTEIN-POLYMER CONJUGATES 3.1. Energetic Balance Between Protein-Polymer Interactions and Polymer Chain Conformation
The phase behavior of peptide-polymer conjugates is dominated by a variety of noncovalent interactions, such as van der Waals interactions, hydrogen bonds, hydrophobic interactions, electrostatic interactions, - interactions, and the entropy associated with polymer chain congurations. A thermodynamically stable conformation arises from the minimization of the overall free energy
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H H H N+ C H R
O C O
b
y dRy
dRx
dRz
Uij
0 r
z r
Entropy of deforming an ideal chain

R 2 + S(N,0) S(N,R ) = 3 k 2 Nb 2
Entropic spring constant 3kT/(Nb 2 ) N: degree of polymerization b: Kuhn length
Figure 3 Energetic considerations of peptide-polymer conjugates. (a) Chemical structure of amino acids, whose side group, R, denes the interactions between each residue pair. (b) Protein sequences, as depicted by the chain of circles of various colors, determine the location and composition of each R group and their relative positions (i.e., interaction-distance relationship). (c) Energetically, it is the combination of multiple pairs of these rather weak interactions that stabilizes individual protein structures and ensures proper protein folding. These secondary interactions, such as van der Waals interactions, electrostatic interactions, and hydrophobic interactions, have strong distance dependences and dissipate quickly as interamino acid distances increase, as shown in the graph that plots energy versus distance. (d ) Schematic representation of the 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 reects a delicate balance between the polymer-residue-solvent interactions and the entropic polymer chain conformation.
Primary structure: sequence of amino acids in a peptide or protein, typically read from the N to C terminus
resulting from all the intra- and intermolecular interactions, and engineering materials with desired properties requires achieving a delicate balance of all these energetic contributions. For natural proteins, the side group, R, of each residue (Figure 3a) denes the interactions between each residue pair that are generally in the range of a few kilocalories per mole. Energetically, it is the combination of multiple pairs of these rather weak interactions that stabilizes individual protein structures and ensures proper protein folding. These secondary interactions have strong distance dependence and dissipate quickly as interamino acid distance increases (Figure 3c). The protein sequence, or primary structure (Figure 3b), essentially determines the location and composition of each R group and their relative positions (i.e., interaction-distance relationship). Weak interactions can be readily strengthened or broken, leading to stimuli responsiveness, and local conditions (such as solvent, pH, and ionic strength) can modulate the conformational properties of the conjugate. To build hybrid biomolecular materials, investigators position synthetic polymers near proteins. The monomer-residue distance is well in the range such that amino acidmonomer
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interactions are equally important as interresidue interactions. The protein cannot distinguish whether the monomer is part of the protein. A similar situation holds for protein-solvent and polymer-solvent interactions (Figure 3d ). Upon attaching polymers, the effects of proteinpolymer interactions on protein structure depend on the chemical nature of the locally surrounding polymer and the spatial distribution of monomers near the protein. In turn, protein-polymer interactions also affect the local polymer chain conformation. Experimental and theoretical studies have shown that the local polymer chain conformation reects a delicate balance between the polymer-residue-solvent interactions and the entropic polymer chain conformation. Within a certain range of molecular weights, attached polymers can arrange locally.
PEG: poly(ethylene glycol) Coiled coil: common tertiary motif, a left-handed supercoil of multiple right-handed -helices
3.2. Fundamental Studies

For protein/peptide-polymer conjugates to reach their full potential as useful materials, the structure and function of the peptide/protein should be maintained upon polymer conjugation. Toward this end, it is important to understand the effect of conjugating polymers on peptide/protein structure, dynamics, and function and the effect of the biomolecule on polymer chain conguration and its materials properties. The effects are dependent on a variety of factors, such as the nature of the polymer, the chemical heterogeneity of the peptide, the site of polymer conjugation, and the solvent media. Success in achieving desirable, functional assemblies relies on understanding the interactions between each building block and delicately balancing and manipulating them to achieve targeted assemblies without interfering with designed structures and functionalities. Such studies, as outlined in Figure 4, have contributed to our understanding of the energetic landscape of peptide-polymer conjugates. 3.2.1. Polymer effect and molecular weight dependence. The most studied peptide-polymer conjugates are of the linear diblock architecture, in which a synthetic polymer is conjugated to a peptide terminus. For example, when poly(ethylene glycol) (PEG) is linked to the end of coiled coils (Figure 4a) (6, 2729), its presence enhances the stability of the peptide structure against temperature and pH changes. However, depending on the length of the peptide sequence and the helical propensity, conjugating the PEG chain to the N terminus reduces the peptide helicity and lowers the degree of association of the bundle (29). This effect is more obvious as the peptides oligomeric 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 restricts the volume available to the polymer chains, leading to molecular crowding and thus favoring lower aggregation states, as well as the unwinding of the ends of the helices. The linear analog is expected to undergo greater loss in peptide structure as the molecular weight of PEG increases. Furthermore, the inuence of the length of PEG on a de novo designed peptide-PEG conjugate, which exhibits a calcium-ion-sensitive transition from random coil to -helix, was investigated: the longer the PEG block, the greater the concentration of Ca2+ necessary to stimulate the coil-tohelix transition. A careful balance of stabilizing and destabilizing effects in the peptide is required to realize rapid responsiveness, and PEGs stabilization of secondary structures is polymer length dependent (31). 3.2.2. Architecture dependence. The architecture of conjugates is another important parameter with which to address fundamental questions. Whereas most conjugates are of the linear diblock variety, 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 coils led to stabilization of peptide secondary and tertiary structures and preservation of built-in
Fundamental studies
Hydrophilic polymer Hydrophobic polymer
a
End conjugate
C, T C, T
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Effect of polymer conjugation Interaction between components
Polymer conformation
Protein conformation & activity
Conjugate conformation
or
0 ns 5 ns 20 ns
Figure 4 Examples of fundamental studies of peptide-polymer conjugates, including (a) hydrophilic end conjugates, (b) hydrophobic end conjugates, (c) hydrophilic side conjugates, and (d ) hydrophobic side conjugates, that address such questions as the effect of polymer conjugation 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 from Reference 29. Copyright 2003 American Chemical Society. Panel b reprinted with permission from Reference 37. Copyright 2010 American Chemical Society. Panel c reproduced from Reference 138 by permission of The Royal Society of Chemistry. Panel d reprinted 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 g reprinted with permission from Reference 49. Copyright 2011 American Chemical Society.
functionality within the interior of the helix bundle. In this case, increasing PEGs length (up to 5,000 Da) increases the helical content of the peptide and enhances its stability against temperature changes. Detailed structural information on the side conjugates was extracted using small-angle X-ray scattering (SAXS) and a newly developed model in which each peptide-PEG conjugate was represented as a Gaussian chain attached to a cylinder, which was further arranged into a bundle-like conguration of multiple cylinders (32). The side conjugates were found to retain helix bundle structure, with the polymers slightly compressed in comparison with the conformation 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 dimension of free chains in solution (R. Lund, J. Shu & T. Xu, unpublished results). Molecular dynamic
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simulations of side conjugates conrmed helix stabilization upon PEG conjugation and showed that the oxygens of PEG interacted favorably with the cationic lysine side chains on the exterior of the helix bundle, which may reasonably account for the compact conguration of the PEG chain revealed by SAXS (33). Such studies show that the conformation of the polymer when conjugated to the peptide is dependent on a balance of the enthalpic interactions between the polymer and the peptide and the entropy of the polymer chain. Similar to the architecture of side conjugates, alternating multiblocks of PEG can be conjugated to peptides through the f position on the heptads by hetero- and homodimeric association of coiled coils. The coiled coils not only retained their capacity to form hetero-oligomers, but also gained the ability to form homo-oligomeric micelles upon PEGylation (34). Even less investigated are peptides containing more than one polymer side chain. A recent study presented the synthesis of peptide-polymer conjugates containing well-dened linear peptide backbones with a dened number of polymeric side chains. Depending on the composition, number, and length of the polymer side chains, the conjugates aggregated to form different topologies (35). In addition, responsive diblock and triblock copolymers of protein-polymer conjugates have been synthesized by grafting polyethyleneglycol methacrylate (PEGMA) and PEGMA-pPEGMA from trypsin, respectively. They exhibited different solution behavior with temperature, dependent on the arrangement of the grafted polymer blocks. Enzyme activity also varied with hybrid architecture, demonstrating the ability to generate conjugates of varied structures, architecture, and solution behavior (36). The great exibility afforded by the conjugate architecture provides a means to tailor the interactions between the components. This also expands the library of available building blocks suitable for a wide array of applications. 3.2.3. Amphiphilicity dependence. The incorporation of hydrophobic moieties to create amphiphilic conjugates can lead to self-assembly of hierarchically structured, biomolecular materials. Systematic understanding of how the peptide structure and functionality are affected upon incorporating hydrophobic moeities is required to direct assemblies in solution, in solid state, and at interfaces. However, depending on the peptide sequence and native structure, studies have shown that the hydrophobic moieties affect peptide structures differently. The attachment of a polystyrene chain to the N termini of a heme-binding coiled coil showed that the presence of hydrophobic polystyrene partially unfolded the peptide secondary structure and consequently compromised the binding pocket within the core of the bundle owing to hydrophobic polymer-peptide interactions at the peptide/polymer interface (Figure 4b) (37). Such effects depend strongly on the periodicity of hydrophobic residues and on interhelix interactions. When covalently linked to polystyrene, Candida antarctica, horseradish peroxidase, and myoglobin showed decreased enzyme activity (Figure 4f ). This was attributed to hydrophobic polystyrene-induced destabilization of the active conformation of each enzyme, disturbed cofactor binding in the apo-protein, or reduced access of the substrate to the active site of the protein (3840). In addition, extensive studies have shown that proteins adsorbed onto hydrophobic surfaces denature and lose their enzymatic activities owing to hydrophobic interactions (4143). A nding observed across many peptide-polymer conjugate systems is that the interfacial zone between the peptide and the synthetic polymer consists of amino acid residues that do not form regular secondary structures (44). Therefore, it may be benecial to vary the architecture of the conjugate or the site of polymer conjugation to retain peptide structure and designed functionality. The effects of the polymers on the peptide structure and function have been systematically investigated as a function of varying degrees of hydrophobicity of the conjugated polymer, showing a direct correlation between the polymer hydrophobicity and the peptide structure in aqueous solution. Researchers studied hydrophobic side conjugates by varying the degree
PMMA: poly(methyl methacrylate)
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PNIPAAM: poly(Nisopropylacrylamide)
of hydrophobicity of the side-conjugated polymer, from polystyrene, PMMA [poly(methyl methacrylate)], to PNIPAAM [poly(N-isopropylacrylamide)]. The presence of the hydrophobic polymer unfolded the peptide helices and induced an -helix to -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 structures were retained by adding organic solvents that solubilized the hydrophobic polymers. These results reiterate that the protein structure in a peptide-polymer conjugate reects competition between the peptide-polymer interactions and the polymer-solvent interactions and is sensitive to the local environment. It is feasible to dissolve amphiphilic peptide-polymer conjugates in organic solvents to enhance their solution processability while maintaining protein structure.
3.3. Challenges
Future progress toward gaining a fundamental understanding of hybrid biomaterials will rely on a combination of the types of studies described above, along with more challenging detailed structural 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 phase behavior of protein-polymer conjugates. One is the polymer chain conformation upon conjugation to 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 of protein structures and polymer segments. In addition to various techniques used to probe protein structures, molecular dynamics simulations or coarse-grained simulations, for example, are needed to provide key insights to design polymers and conjugates. PEGs interaction with proteins has been investigated to determine its conformation when conjugated 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 (4547). Studies that indicate reduced immunogenicity of proteins upon PEGylation assume a shroud model. A shroud model was also presumed in the analysis of a size-exclusion chromatography study (45) and a SAXS study of PEG-antibody conjugates, in which PEG is thought to effectively cover part or all of the antibody (46). These observations support the view that PEG interacts with proteins to form an exterior shell. In an alternate model, however, PEG does not interact signicantly with the protein. Rather, the conjugate forms a dumbbell-like architecture, in which two noninteracting entities are covalently linked (47, 48). In recent small-angle scattering studies that investigated the conguration of PEG conjugated to lysozyme, human growth hormone, or human galectin-2, a dumbbell conguration was observed, with Rg of conjugated PEG similar or greater to that when free in solution (47, 48). Furthermore, a model that combines the dumbbell and shroud views was observed in SAXS studies of PEG-hemoglobin (48) and coiled-coil-PEG side conjugates. These conicting observations may result from the properties of a PEG-protein conjugate depending on a variety of factors, such as the site of conjugation, the molecular weight of PEG, the number of PEG chains per protein, and the chemical heterogeneity of the surface of the protein. Furthermore, extracting such information from an experimental point of view is challenging, even with scattering techniques, and clear conclusions may be difcult to make. Computer simulations are vital in garnering information unobtainable from experiment alone and for interpretation of experimental results. Molecular dynamic simulations (Figure 4g) may provide a means to determine the local arrangements of segments from proteins and polymers that cannot be readily extracted experimentally (49, 50). Simulations, in conjunction with systematic experimental studies as described above, provide a means to address fundamental issues that will contribute to the rational design of these hybrid materials.
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Stimuli-responsiveness
a
Acid pH (<4)
pH Neutral pH (5<pH<9) Basic pH (>10)
Enzyme
pH
c
Ion/cofactor binding
Temperature
Trypsin conjugate hybrid I Trypsin conjugate hybrid II
Ca2+
Ca2+
Peptide = random coil

Ca2+
Peptide = -helix T < LCST Hydrophobic Hydrophilic T > LCST T < LCST T > LCST
Random coil
T < LCST -helix
T > LCST
T < LCST
T > LCST
Ca2+
Figure 5 Responsiveness of peptide polymer conjugates to various stimuli, including changes in (a) pH, (b) enzymatic activity, (c) ion/cofactor binding, 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 Hans Borner. 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 and strategies to direct polymeric nanostructures, while achieving structural control at the atomic level that has been elusive with synthetic polymers alone. Various functions, both natural and nonnatural, can be incorporated into the peptide/protein block through sequence design and modication, and the great monomer selection available for the synthetic block provides the ability to mediate the interactions between the peptide and its environment and to control the self-assembled structure in a predetermined way. Furthermore, the peptides responsiveness to external 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 when desired (Figure 5).
4.1. Solution
Polypeptides: peptides of simple sequences typically comprising one or a few amino acid species
Engineering amphiphilicity into peptide-polymer conjugates enables them to self-assemble into functional, biomolecular nanostructures, such as spherical micelles, cylindrical micelles, and vesicles, when dissolved in a selective solvent for one block (3, 8, 9, 44, 5356). A majority of studies have focused on conjugates comprising hydrophobic synthetic polymers and water-soluble polypeptide segments that fold into -helices (44, 53, 5759). These form a multitude of structures with tunable morphologies (6062) and stimuli responsiveness (58, 6365). In addition, -sheet-forming conjugates form brils directed by peptide organization, with a peptide core and a polymer corona (66), that can reach micrometers in length and display a superhelical ne structure with a lefthanded helical twist (67, 68). With increased peptide complexity, a heterodimer coiled coil was used to noncovalently link PEG and polystyrene blocks, and the resultant amphiphilic PEG-peptidepolystyrene triblock copolymer assembled into thermoresponsive micelles that transformed from a rodlike to spherical structure upon heating (69). In another report, a thermoresponsive triblock comprising poly(diethylene glycol methyl ether methacrylate) conjugated to the ends of a triple-helix-forming collagen-like peptides displayed a transition from spherical aggregates that increased in size with increasing temperature up to 65 C to brils at 75 C (70). Giant amphiphiles, compounds comprising an entire enzyme or protein as the head group and a hydrophobic polymeric tail attached to a preselected, well-dened location, are also able to form typical structures in solution (3840, 7181). In addition, triblock hybrid copolymer analogs have been synthesized and were 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 monolayers of tripeptide-polystyrene diblocks and peptide-polystyrene-peptide amphiphiles were investigated at the air-water interface (84). The architectural variations had a great impact on the self-assembly of the amphiphiles, especially for the transition from the gas phase to the liquid phase, because steric interactions between polymer chains are dominant. However, in a condensed phase, the packing of the various architectures was comparable (84). A -sheet-poly(nbutyl acrylate) conjugate spread at the air-water interface showed a -sheet network that consists of antiparallel -sheets oriented parallel to the interface. Stable monolayers with low compressibilities formed when -sheet assembly occurred in monolayers of close-packed conjugates. The Langmuir-Schaefer transfer of the monolayer seeded with stearic acid resulted in a monolayer containing 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(Lalanine) hydrophobic graft chains demonstrated strong pH-dependent assembly. In acidic conditions, the protonated allylamine moieties dissolved in the aqueous phase, while the hydrophobic grafts acted as anchors to keep the monolayer at the interface. In alkaline conditions, the polymer was located entirely at the interface, forming stable and closely packed uniform monolayers owing to dehydration of the alkylamine moieties, with the graft chains forming -sheet domains (86). -Helix-forming polypeptide-PEG conjugates spread at the air-water interface were also found to form stable monolayers, with the polypeptide forming -helices that were oriented slightly normal to the interface at high surface pressures. Langmuir-Blodgett monolayers deposited on solid substrates conrmed that the -helix of the peptide block was oriented slightly normal to the surface, although the presence of PEG tilted the rod at the interface, preventing an entirely upright orientation (87).
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4.3. Bulk
Typical linear block copolymer (BCP)-type homopolypeptide-polymer conjugates, in which the peptide forms simple secondary structures, self-assemble into morphologies similar to those seen with fully synthetic BCPs (5, 10, 58, 69). Most -helical polypeptide-polymer conjugates tend to form hexagonal-in-zigzag lamellar morphologies, with the peptide helices either interdigitated or folded and packed hexagonally (53, 58, 59, 8894). Hexagon-in-hexagonal morphologies have also been observed (58). -sheet peptide-PEG conjugates have been found to form lamellar structures with lyotropic liquid crystal nematic and hexagonal columnar phases and PEG crystallization (95, 96). Similar to the case in solution, the -sheet-forming peptide has a signicant impact on the solid-state structure of the resulting conjugate. Longer PEG spacers lowered the tendency to form antiparallel -sheets and resulted in microphase separation (97). With larger biomolecules, protein-PNIPAAM conjugates formed highly disordered lamellae or hexagonally perforated lamellae. Solvent annealing resulted in a transition toward lamellar nanostructures with proteins packed in a bilayer and improvements in long-range order (98).
BCP: block copolymer
4.4. Thin Film

Similar to that observed in bulk, -helical polypeptide-polymer conjugates also form hexagonalin-lamellar morphologies in thin lms. A recent study presented peptide-polymer conjugates containing well-dened linear peptide backbones with a dened number of polystyrene or PNIPAAM side chains. Spin-coated peptide-polystyrene conjugates formed honeycomb structures for small polymer chains and coral-like morphologies for longer side chains, whereas peptide-PNIPAAM conjugates aggregated to ring-shaped topologies (35). A thin lm of a triblock containing a conjugated polymer anked by two polypeptide blocks retained electroactive and photoactive properties of the polymer. The presence of the polypeptide blocks did not impede charge injection, transport, and photophysical processes essential for electroluminescence. Different nanostructure assemblies were observed depending on the copolymer composition and the secondary structure of the polypeptide block (99). In addition, peptide/protein-polymer conjugates can be coassembled with synthetic BCPs to create composites that exhibit hierarchical structural complexity and chemical functionality localized to specic microdomains of the BCP thin lm. Ferritin-PEG nanoparticles were incorporated in a poly(2-vinylpyridine)-b-poly(ethylene glycol) thin lm. The resulting composite contained ferritin-PEG nanoparticles conned to PEG microdomains, showing that direction of protein localization within BCP lms is possible. Recently, the simultaneous coassembly of PS-b-PEO with heme-binding coiled-coil-PEG side conjugate and myoglobin-PEG conjugates was reported to form hierarchically structured lms using organic solvent-based solution processing (Figure 6) (100). Protein structure was retained, heme-binding by helix bundles was reconstituted, and the retention of myoglobin peroxidase activity was observed in thin lms. The hierarchical organization of these multiple functional elements, through a combination of protein folding, biomolecular recognition, and BCP microphase separation, represents a new approach to fabricate functional biomolecular materials using peptides and enzymatically active proteins. This work demonstrates the feasibility of synchronizing multiple self-assembly processes to achieve hierarchically structured, protein-containing soft materials with molecular-level control. Conjugates have also been adsorbed onto surfaces. BCPs containing a polypeptide segment with alkyl side chains hierarchically self-assemble on graphite through epitaxial adsorption of the peptide segment. Morphologies were concentration dependent, ranging from island-like aggregates, monolayers, and monolayers with larger nanorods to monolayers with ring-shaped
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aggregates as the concentration increased. These diverse morphologies were a result of hierarchical assembly induced from peptide -helix folding, epitaxial adsorption, rod-coil contrast, and amphiphilicity (101). The various nanostructures formed by self-assembly provide tremendous promise for biomedical and nonbiological applications. These studies, in conjunction with fundamental studies, provide design principles on which to develop tailored nanostructures with the desired properties to suit specic 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 stimuli responsiveness make them amenable to such applications, as they can easily interface with the biological and synthetic worlds.
5.1. Drug/Gene Delivery

An immense amount of work has been done to generate micelles based on polypeptide-PEG conjugates for small-molecule anticancer drug and gene delivery. Polypeptide-PEG micelles are generally tens of nanometers in size with various intermolecular interactions in the core-forming polypeptide block to regulate micelle stability for prolonged circulation and controlled drug release (102109). Polypeptides such as poly(aspartate), poly(glutamate), and poly(lysine) have been utilized. Common anticancer drugs can be incorporated into the core of the micelle through hydrophobic interactions or covalent attachment to polypeptide side chains. Micelles show prolonged blood circulation in comparison to free drugs by avoiding glomerular ltration and reticuloendothelial system uptake and effectively accumulating in the tumor via the enhanced permeation and retention effect. Micelles typically demonstrate higher efcacy with fewer side effects than free drugs as well. For the delivery of nucleic acids, one usually employs a polyion complexation between anionically charged DNA or RNA and a BCP having a hydrophilic segment and a cationic segment, such as poly(lysine). The polyplex micelles exhibit efcient gene introduction in cultured cells and also show gene expression in vivo. Stimuli-responsive micelles have also been generated to release cargo at target sites when desired based on environmental cues. For example, differences in the concentration of reductive agents between the extracellular and intracellular environment and pH reduction in endosomes can be used as triggers for drug release to achieve smart nanocarriers. In addition, endosomal escape and cytosolic delivery are especially important for effective delivery. Investigators used the fusogenic activity of a pH-sensitive micelle, PEG-poly(histidine), in endosomes to facilitate the
Figure 6 Examples 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 lms. Panel a reprinted with permission from Reference 140, copyright 2003 Springer, and reprinted with permission from Reference 60, copyright 2005 American Chemical Society. Panel b reprinted with permission from Reference 97, copyright 2010 American Chemical Society; reprinted with permission from Reference 91, copyright 2002 Elsevier; and reprinted with permission from Reference 59, copyright 2000 American Chemical Society. Panel c reproduced from Reference 84 by permission of The Royal Society of Chemistry and reprinted with permission from Reference 85, copyright 2008 American Chemical Society. Panel d reproduced 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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cytosolic delivery of cargo. Furthermore, specic ligands, such as antibodies, transferrin, folate, sugars, and peptides, can be appended to the surface of the micelle to achieve active targeting to cancer cells for increased tumor accumulation, increased cellular uptake, and increased transfection efciency in vitro. Despite these many advances, it remains a signicant challenge to generate organic nanocarriers with a long circulation half-life, effective tumor penetration, and efcient clearance of metabolites. A new family of amphiphiles based on coiled-coil 3-helix bundle forming peptide-PEG conjugates has recently been developed. The amphiphiles self-assemble into monodisperse micellar nanoparticles, 15 nm in diameter (110). By incorporating unique protein tertiary structures in the head group of an amphiphile, investigators can generate organic nanoparticles with tunable stability, ligand clustering, and controlled disassembly to meet current demands in nanomedicine. Cargo can also be attached to a polymer backbone via coiled-coil linkers formed by the heterodimerization of two complementary peptide sequences that are linked to the polymer carrier and the cargo, respectively (28, 111). Not only are the coiled-coil linkers useful for binding and releasing cargo, but they may also play an active role in enhancing and directing intracellular transport and trafcking (111, 112). Rather than serving solely a structural function in drug-delivery systems, peptides can also serve a therapeutic function. For example, a micellar nanocontainer delivery and release system was generated based on -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 disassembly and release of peptides in unaggregated forms. This demonstrates the potential in the targeted delivery of peptides for therapeutics applications (52). Conjugates containing coiled coils as a therapeutic molecular switch have also been developed. PEG-FosW was able to form a stable coiled-coil heterodimer with the target c-Jun sequence of the oncogenic AP-1 transcription factor (112).
HPMA: N-(2-hydroxypropyl) methacrylamide
5.2. Protein Therapeutics

The most commonly used polymer in hybrid biomaterials is PEG. PEGylation has proven to be an effective strategy to enhance the kinetic stabilization of proteins, to obtain catalytic activity at very high temperatures, and to improve the stability, pharmacokinetics, and biodistribution of therapeutic proteins (2, 113, 114). FDA-approved PEGylated proteins include Adagen, Oncaspar, Krystexxa, PEGASYS, and PEG-INTRON (115). The unique chemical properties of PEG, such as its high solubility, amphiphilicity, and inertness, are key to its effectiveness (2, 116118). Most studies have focused on the improved pharmacological performance of PEGylated proteins compared with their unmodied counterparts and their differences in bioactivity. Variables such as the molecular weight of PEG, the number of PEG chains per protein, the architecture of the polymer (branched or linear), and its site of conjugation can affect the in vivo half-life and the activity of proteins. Signicant work has also been done with protein-HPMA [N(2-hydroxypropyl)methacrylamide] conjugates. HPMA is a hydrophilic, biocompatible polymer
Figure 7 Examples 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 permission from Reference 110, copyright 2012 American Chemical Society. Panel c reproduced from Reference 144 by permission of the Nature Publishing Group. Panel d reprinted with permission from Reference 141, copyright 2009 Wiley, and reprinted with permission from Reference 145, copyright 2011 Elsevier.
whose -carbon substitution and N-substituted amide bond ensure the hydrolytic stability of the side chains. Results are similar to those garnered with PEG (119122). Thermoreversible protein-PNIPAAM conjugates have also been well studied. Similar to PEG-ylated and HPMA analogs, PNIPAAM conjugates exhibit retention of activity, even higher than the native enzyme, and improved thermal stability (123, 124). However, they can also be separated from solution by thermal precipitation. Streptavidin-PNIPAAM conjugates can reversibly block biotin association as the polymers conformation changes at its lower critical solution temperature, or be engineered to repeatedly bind biotin at room temperature and release bound biotin at the lower critical solution temperature as the temperature is cycled (125, 126). Polymer conjugation to proteins has made a large impact on protein therapeutics and in generating stimuli-responsive biomolecules with tailored activities and solubilities.
5.3. Nonbiological Applications

Aside from the typical biomedical applications usually associated with peptide-polymer conjugates, these materials are also capable of impacting nonbiological applications, ranging from gas separation to optoelectronics and catalysis (Figure 8). For instance, peptide-polymer conjugates can be used to generate porous thin lms containing subnanometer channels oriented normal to the surface, which exhibit unique transport and separation properties and can serve as selective membranes for separation and protective coatings. A new approach has been developed in which the growth of cyclic peptide nanotubes can be directed in a structural framework set by the self-assembly of BCPs (127). By conjugating polymers to cyclic peptides, the subunit of an organic nanotube can be selectively solubilized in one copolymer microdomain. The conjugated polymers also mediate interactions between nanotubes and the local medium and guide the growth of nanotubes in a conned geometry. This led to subnanometer porous membranes containing high-density arrays of through channels. This new strategy takes full advantage of the nanoscopic assembly of BCPs and the reversibility of organic nanotube growth and circumvents impediments associated with aligning and organizing high-aspect-ratio nano-objects normal to the surface. Oligopeptide-oligothiphene hybrids, which form nanostructured brillar aggregates, are promising for optoelectronic applications because of the self-assembly and stimuli responsiveness provided 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-ordered optoelectronic segments by the self-assembly of biological moieties, to eventually generate function by the structuring of materials. The photophysical changes observed upon assembly highlight the strong electronic communication within the amyloid-like aggregates, which are mediated by -stacking among molecular components, a critical component necessary for charge transport or exciton delocalization in nanostructures of semiconductive -systems (130). Fibrillar superstructures are promising if combined with existing top-down technologies, such as plotting or printing processes, because this should allow for a rational design of macroscopic materials with anisotropic optoelectronic properties and hierarchically ordered ne structures (68). Furthermore, researchers have achieved the macroscopic alignment of peptides bearing -conjugated functionality via a solution method to process self-assembling peptide amphiphiles into highly aligned macrostructures (hydrogel noodle formation technique) (131), which results in materials displaying unique photophysical and anisotropic electrical responses. The resulting hydrogel noodles resulted in alignment of the -electron oligomers and led to global directionality among the internal -stacked chromophores. The anisotropic electrical properties of the organic semiconductors buried within the noodles led to an increase of an order of magnitude in hole mobilities along the length of the macrostructure (132). In addition, a
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Figure 8 Examples 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 American Chemical Society. Panel b reprinted with permission from Reference 129, copyright 2008 Wiley, and reprinted with permission from Reference 130, copyright 2008 American Chemical Society. Panel c reprinted with permission from Reference 142. Panel d (directed mineralization) reprinted with permission from Reference 143, copyright 2008 Wiley, and (nanoparticle growth) reproduced from Reference 134 by permission of The Royal Society of Chemistry.
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polypeptide-oligothiophene conjugate, which formed organogels with the peptide adopting an -helical conformation, was used to fabricate organic photovoltaic and organic eld-effect transistor devices by deposition of the PCBM-blended active layer at concentrations shown to induce self-assembly of the polymer. These devices showed signicantly greater hole mobility, short circuit current, and efciency compared with control compounds. This work establishes the potential of these hybrid materials to increase device performance (133). Investigators achieved the controlled formation of silver nanoparticle arrays with high particle density and short interparticle spacing by using supramolecular nanotapes of oligopeptide-PEG conjugates as templates to direct the nucleation and growth of silver nanoparticles. The optical and electronic properties of nanoparticles make them desirable for applications such as light trapping in solar cells, which require control of their organization. The hierarchical organization provided by the oligopeptides allows for organization of nanoparticles at smaller length scales than those currently achieved with synthetic BCPs alone (134). Finally, vesicles made from protein-polymer conjugates can be used to generate bionanoreactors for catalysis. Vesicles comprising HRP-PS or HRP-PMMA encapsulating GOx were shown to undergo cascade reactions, in which GOx oxidized glucose to form H2 O2 , which was subsequently used by HRP to oxidize ABTS (135). A nanocontainer was also formed from atom transfer radical polymerizationmediated in situ preparation of BSA-PS giant amphiphiles. A one-pot hierarchical incorporation of enzymes was achieved without interfering with the protein integrity of the overall aggregate. The nanocontainers are permeable and can be used as nanoreactors that display catalytic activity (136). Although biomedical applications of peptide-polymer conjugates are more thoroughly studied in comparison to nonbiological applications, the large library of proteins found in nature provides immense opportunities for the practical utilization of various functions, and their hierarchical organization allows for the generation of nanostructures with an order 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 materials with a wide range of applications. Because these conjugates are hybrid materials that combine the biological and synthetic worlds, research in this area often lies at the interface between many disciplines. Such work ranges from fundamental materials science and polymer physics to chemical biology, structural characterization in real and reciprocal spaces, and cell biology. From a materials science point of view, a fundamental understanding of these hybrids materials is necessary, along with control over their self-assembly into well-dened nanostructures, if these building blocks are to reach their full potential in biological and nonbiological applications. Because many noncovalent 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 assemblies. Detailed structural characterization is also necessary to fully deduce the structure of the peptide and the polymer chain conformation. With fundamental structural and behavioral properties understood, the knowledge gained can then be used to rationally design peptide-polymer conjugates with desired properties. Many parameters can be tailored to achieve a target, including the 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 the conjugate). When applying these materials to applications, it is important to engineer well-dened materials whose structure and behavior are well characterized. This will aid in understanding and tailoring the behavior of these materials in various environments. Toward this end, scattering is 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 future progress. The complexity and diversity of protein/peptide-polymer conjugates are obvious. Computation and simulation are in critical need to provide valuable guidance toward molecular design and to develop basic understanding in their phase behavior, not only in their static 3D structures, 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 afliations, memberships, funding, or nancial holdings that might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We acknowledge support from the Ofce of Science, Ofce of Basic Energy Sciences, of the US Department of Energy under contract DE-AC02-05CH11231 ( J.Y.S. and T.X.) and the Ofce of the 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 gures for republication in this article. LITERATURE CITED
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Contents
The Hydrogen Games and Other Adventures in Chemistry Richard N. Zare p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Annual Review of Physical Chemistry Volume 64, 2013
Once upon Anion: A Tale of Photodetachment W. Carl Lineberger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 21 Small-Angle X-Ray Scattering on Biological Macromolecules and Nanocomposites in Solution Clement E. Blanchet and Dmitri I. Svergun p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 37 Fluctuations and Relaxation Dynamics of Liquid Water Revealed by Linear and Nonlinear Spectroscopy Takuma Yagasaki and Shinji Saito p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 55 Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy Chao-Yu Chung, John Boik, and Eric O. Potma p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 77 Multidimensional Attosecond Resonant X-Ray Spectroscopy of Molecules: Lessons from the Optical Regime Shaul Mukamel, Daniel Healion, Yu Zhang, and Jason D. Biggs p p p p p p p p p p p p p p p p p p p p p p 101 Phase-Sensitive Sum-Frequency Spectroscopy Y.R. Shen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 129 Molecular Recognition and Ligand Association Riccardo Baron and J. Andrew McCammon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 151 Heterogeneity in Single-Molecule Observables in the Study of Supercooled Liquids Laura J. Kaufman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 177 Biofuels Combustion Charles K. Westbrook p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 201 Charge Transport at the Metal-Organic Interface Shaowei Chen, Zhenhuan Zhao, and Hong Liu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221 Ultrafast Photochemistry in Liquids Arnulf Rosspeintner, Bernhard Lang, and Eric Vauthey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 247
Cosolvent Effects on Protein Stability Deepak R. Canchi and Angel E. Garc a p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 273 Discovering Mountain Passes via Torchlight: Methods for the Denition of Reaction Coordinates and Pathways in Complex Macromolecular Reactions Mary A. Rohrdanz, Wenwei Zheng, and Cecilia Clementi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 295 Water Interfaces, Solvation, and Spectroscopy Phillip L. Geissler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 317 Simulation and Theory of Ions at Atmospherically Relevant Aqueous Liquid-Air Interfaces Douglas J. Tobias, Abraham C. Stern, Marcel D. Baer, Yan Levin, and Christopher J. Mundy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 339 Recent Advances in Singlet Fission Millicent B. Smith and Josef Michl p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 361 Ring-Polymer Molecular Dynamics: Quantum Effects in Chemical Dynamics from Classical Trajectories in an Extended Phase Space Scott Habershon, David E. Manolopoulos, Thomas E. Markland, and Thomas F. Miller III p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 387 Molecular Imaging Using X-Ray Free-Electron Lasers Anton Barty, Jochen Kupper, and Henry N. Chapman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 415 Shedding New Light on Retinal Protein Photochemistry Amir Wand, Itay Gdor, Jingyi Zhu, Mordechai Sheves, and Sanford Ruhman p p p p p p p p 437 Single-Molecule Fluorescence Imaging in Living Cells Tie Xia, Nan Li, and Xiaohong Fang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 459 Chemical Aspects of the Extractive Methods of Ambient Ionization Mass Spectrometry Abraham K. Badu-Tawiah, Livia S. Eberlin, Zheng Ouyang, and R. Graham Cooks p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 481 Dynamic Nuclear Polarization Methods in Solids and Solutions to Explore Membrane Proteins and Membrane Systems Chi-Yuan Cheng and Songi Han p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 507 Hydrated Interfacial Ions and Electrons Bernd Abel p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 533 Accurate First Principles Model Potentials for Intermolecular Interactions Mark S. Gordon, Quentin A. Smith, Peng Xu, and Lyudmila V. Slipchenko p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 553
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Structure and Dynamics of Interfacial Water Studied by Heterodyne-Detected Vibrational Sum-Frequency Generation Satoshi Nihonyanagi, Jahur A. Mondal, Shoichi Yamaguchi, and Tahei Tahara p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 579 Molecular Switches and Motors on Surfaces Bala Krishna Pathem, Shelley A. Claridge, Yue Bing Zheng, and Paul S. Weiss p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 605 Peptide-Polymer Conjugates: From Fundamental Science to Application Jessica Y. Shu, Brian Panganiban, and Ting Xu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 631
Indexes Cumulative Index of Contributing Authors, Volumes 6064 p p p p p p p p p p p p p p p p p p p p p p p p p p p 659 Cumulative Index of Article Titles, Volumes 6064 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 662 Errata An online log of corrections to Annual Review of Physical Chemistry articles may be found at http://physchem.annualreviews.org/errata.shtml
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ANNUAL REVIEWS

Peptide-Polymer Conjugates: From Fundamental Science to Application

Nonbiological applications Peptidepolymer conjugates

Directed biomineralization Immunology

2. SYNTHESIS OF PEPTIDE/PROTEIN-POLYMER CONJUGATES

Methods of peptide synthesis

Solid-phase peptide synthesis

Coupling of next amino acid Designed polypeptide X N H Y2 R2 H O N

Native chemical ligation

Polymer-peptide conjugate X Cu2+/L + P n kp Monomer PnS S Pm kadd k add PnS S + kt

Intramolecular S-N acyl shift H O N Peptide A Peptide B O SH

kp Monomer H3 C CH3 CH3 CH3 N O CH3

CH3 CH3 CH3 N O CH3 H3 C n O O CH3 O CH3 F N H3 C H H3C

2.1. Peptide/Protein Synthesis

2.2. Polymer Synthesis and Conjugation

2.3. Challenges: Synthesis and Characterization

Entropy of deforming an ideal chain

Entropic spring constant 3kT/(Nb 2 ) N: degree of polymerization b: Kuhn length

3.2. Fundamental Studies

Effect of polymer conjugation Interaction between components

Protein conformation & activity

PMMA: poly(methyl methacrylate)

pH Neutral pH (5<pH<9) Basic pH (>10)

Peptide = random coil

T < LCST -helix

4. TOWARD FUNCTIONAL HYBRID BIOMATERIALS: SELF-ASSEMBLY

4.2. Polar/Nonpolar Interfaces

4.4. Thin Film

Side view d=5 z Rcore ~2.5 nm y x d = 16

Solvent In water Copolymer HOPG Low High concentration concentration

5. TOWARD FUNCTIONAL HYBRID BIOMATERIALS: EMERGING APPLICATIONS

5.1. Drug/Gene Delivery

HPMA: N-(2-hydroxypropyl) methacrylamide

5.2. Protein Therapeutics

5.3. Nonbiological Applications

Blend 8CP-polymer conjugates with BCP

Grow 8CP nanotubes within BCP microdomains

Cyclic peptide (8CP) Polymer conjugated to 8CP Block copolymer (BCP)

Directed mineralization/nanoparticle growth Top view

Peptide -sheet core

www.annualreviews.org Peptide-Polymer Conjugates

6. PERSPECTIVES AND OUTLOOK

www.annualreviews.org Peptide-Polymer Conjugates

Annual Review of Physical Chemistry Volume 64, 2013

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