Computational Design of Four-Helix Bundle Proteins That Bind NonbiologicalCofactors

Andreas Lehmann and Jeffery G. Saven*

Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104

Recent work is discussed concerning the computational design of four-helix bundle proteinsthat form complexes with nonbiological cofactors. Given that often there are no suitable naturalproteins to provide starting points in the creation of such nonbiological systems, computationaldesign is well suited for the design and study of new protein-cofactor complexes. Recent designefforts are presented in the context of prior work on the de novo design and engineering ofporphyrin-binding four-helix bundle proteins and current developments in nonlinear opticalmaterials. Such protein-nonbiological cofactor complexes stand to enable new applications inprotein science and materials research.

Introduction

The well-defined, highly specific properties of biomoleculesat the atomic scale are the basis for many advances inbiotechnology and provide the foundation for the formation ofhigher-order structures often found in biology. Proteins and otherbiomolecules are potentially cost-effective molecular materialsthat are rapidly realized and largely environmentally benign.Consequently, the design of protein structure and function hasreceived much interest, usually with two goals in mind: to betterunderstand the molecular features and interactions that determinestructure and functionality and to apply this knowledge to thedesign of new functional proteins and biomaterials. Furtherdevelopment of such materials is expected to lead to newapplications in nanotechnology, tissue engineering, drug deliv-ery, biosensing, medical devices, implants, and regenerativemedicine (1-7). Especially appealing for these applications isthe potential of proteins to form “smart” materials, which aresensitive to stimuli such as changes in pH, ionic strength,temperature, light, oxidation/reduction state, concentration, orenzymatic acivity (2, 3, 8). Proteins display an immenseversatility of three-dimensional structures and functions, featuresthat are determined (for most proteins) solely by the proteinsequence (9). Comprising the 20 natural amino acids with theirhydrophobic, polar, aromatic, and ionizable side chains, proteinsfold into structures that allow for a wide array of biochemicaland biophysical functions, e.g., cytoskeleton assembly, transport,signaling, bioenergetics, metabolism, and regulation. Comparedto the challenge of accurately predicting structure and functionsolely from sequence information (10, 11), experimentallyrealizing new proteins is comparatively straightforward usingin vitro peptide synthesis or in vivo overexpression. Theselection of mutations or new sequences, however, is oftennontrivial, given the many interactions involved and the largenumbers of possible variants. The ease of sequence modificationfacilitates the use of the self-organizing properties of proteinsystems to yield new systems outside of what has evolvednaturally, e.g., through the use of non-canonical amino acids(12-14).

Alternatively, nature overcomes the physicochemical limita-tions of the natural amino acids through the incorporation ofcofactors, which frequently provide the structure and electronicproperties required for electro-optical or catalytic activity. Suchcofactors take the form of metal ions, organometallic com-pounds, and inorganic (15) or organic molecules. Thus, apossible way to expand the functional properties of proteins isto create systems containing nonbiological cofactors, i.e.,cofactors with no precedents in natural systems. Nonbiologicalcofactor-protein complexes may lead to the development ofnew materials that draw from developments in both molecularbiology and nonlinear optical materials.

Complexes of de novo designed proteins with such nonbio-logical cofactors are the subject herein, and specifically systemsinvolving nonbiological cofactors with metal porphyrin sub-groups. Several de novo designed proteins that bind naturalporphyrin cofactors are discussed. Nonbiological cofactors thatcontain metal porphyrins are presented as promising nonlinearoptical chromophores with possible optoelectronic applications.Last, the computational design of protein-cofactor systems isdiscussed.

Design of Heme-Binding Proteins

The heme molecule, iron protoporphyrin IX, is a ubiquitouscofactor that occurs in many natural proteins. There are currentlyover 200 non-redundant structures of heme-containing proteinsdeposited in the Protein Data Bank (16, 17). Heme proteins arefunctionally and structurally diverse and play roles in electrontransfer, substrate oxidation, metal ion storage, ligand sensing,transport, and gene expression (18-20). Porphyrinic cofactorssuch as hemes and chlorophylls are also essential in fundamentalbiological energy conversion processes such as photosynthesisand cellular respiration (21). These and other tetrapyrroliccompounds have been called the “pigments of life” (22-25).The versatility of heme- and other porphyrin-containing proteinsdraws upon several features: the delocalizedπ-conjugatedporphyrin macrocycle, the multiple oxidation states of the centralmetal ion, the amino acid residues that coordinate the metalion, and the residues near the cofactor. In heme, the Fe ion iscoordinated by four equatorial nitrogen atoms in the porphyrinmacrocycle, and binding to the protein is realized through amino

* To whom correspondence should be addressed. Tel:+001 (215)573-6062. Fax:+001 (215) 573-2112. E-mail: saven@sas.upenn.edu.

74 Biotechnol. Prog. 2008, 24, 74−79

10.1021/bp070178q CCC: $40.75 © 2008 American Chemical Society and American Institute of Chemical EngineersPublished on Web 01/16/2008

acid side chains. Typical axial coordination atoms are N(ε) ofHis residues or S of Met residues. The Fe ion can bepentacoordinated or hexacoordinated. Five-point coordinationis usually based on His ligands, and six-point coordination isfrequently bis-His- or His-Met-based, although His-Thr coor-dination is also observed (20). Electronic properties of thecofactor-protein complex may also be influenced by residuesnear the cofactor that are not involved in metal coordination,and the local cofactor environment within the protein oftencomprises hydrophobic residues. The midpoint reduction po-tential Em is often used to quantify the influence of the localcofactor environment on redox properties.Em can vary greatlywithin heme proteins, ranging from-550 to+395 mV (relativeto the standard hydrogen electrode, SHE) (26, 27). Dependingon the particular heme protein environment, both the coordina-tion chemistry and the local cofactor environment can influenceEm (20).

Some natural heme proteins facilitate highly organizedbiochemical events. One example is the bovine transmembranecytochromebc1 complex (28), which is part of the oxygenrespiratory chain and catalyzes the reduction of cytochromecusing the proton-motive quinone(Q)-cycle, combining a trans-membrane proton pump with electronic charge transfer acrossprotein subunits (29, 30). The complex is composed of twob-hemes, onec-heme, a [2Fe-2S] Rieske protein, and twoquinones bound within its 11 protein subunits. Growing out ofa desire to study these complex interactions, protein maquetteshave been developed as simplified models of natural proteins(31, 32), so as to establish minimal requirements for proteinfunction and to facilitate the study of subprocesses associatedwith the larger complex. Robertson et al. (32) first reported therational de novo design of a synthetic four-helix bundlemultiheme protein, which mimicked a four-helix bundle at thecenter of the cytochromeb subunit of the cytochromebc1

complex. In this design, four heme groups were bound in theinterior of the bundle, demonstrating that multiheme redoxcenters could indeed be transferred into protein maquettes.Similar efforts examined specific interactions in the proteininterior (33) introducing Zn- (34, 35) and heme-binding (36)sites into the designed bundles. However, the first maquettespossessed a somewhat disordered, conformationally nonspecific(non-native) hydrophobic core (37), which motivated subsequentiteratively improved designs (38, 39). Recently, a rationallydesignedD2-symmetrical tetrameric di-heme four-helix bundleprotein has been realized, for which NMR data suggest a uniquewell-folded structure (well-resolved15N HSQC and13C HSQCNMR resonances) (40). Other approaches have also led tosuccesses in the design of new heme-binding proteins. Rau etal. (41) reported the design of a template-assisted syntheticpeptide (TASP (42)), which formed a four-helix bundle thatbound two hemes at its two bis-His coordination sites. In thispeptide, bundle formation was supported through covalentlylinking each of the helices to a cyclic decapeptide. Anotherdesign approach was taken by Hecht and co-workers, whodevised a hybrid strategy between rational design and combi-natorial library-based protein engineering by constraining com-binatorial libraries to simple binary-patterned sequences of polarand nonpolar residues. The authors showed that this strategy issufficient to construct four-helix bundle proteins (43-46), someof which exhibit heme-binding properties (44). These recentsuccesses with identifying novel heme-containing proteinssuggest that crafting more complicated proteins containingnonbiological cofactors is within reach.

Nonbiological Cofactors as Nonlinear OpticalMaterials

Nonlinear optical (NLO) materials remain an active andimportant area of research (47). NLO materials are relevant tofiber optic networks, optical computing, laser development, andother applications where NLO properties can be used to controlthe direction, frequency, amplitude, phase, and polarization (47-53) of light as it propagates through a material. With theemergence of photonic technologies in areas such as telecom-munications, there is a strong technological demand for high-performance NLO materials (47).

When light passes through an optical medium, the electricfield E is attenuated by the dynamical polarizationP of themedium that responds linearly and nonlinearly to the externallyapplied field. Assuming that the polarization is weak comparedto the binding forces between electrons and nuclei,P may beapproximated using a series expansion in terms of susceptibilitiesø(n) of the bulk material:

Here, ø(1) is the linear susceptibility tensor, describing theproportionality ofP andE. ø(1) is a second-rank tensor, andø(n)

for n g2 arenth-order nonlinear susceptibilities;ø(2) is a third-rank tensor, andø(3) is a fourth-rank tensor. Susceptibilities ofinterest for NLO materials are investigated primarily forn ) 2and 3. The susceptibilitiesø(1), ø(2), andø(3) are bulk propertiesbut have molecular analogs in the single-molecule linearpolarizability R, and the first and second (nonlinear) hyperpo-larizabilitiesâ andγ, respectively. It can be shown that all even-ordered susceptibilities (e.g.,ø(2)) are zero if the material isisotropic. Similarly, the corresponding hyperpolarizabilityâ iszero in molecules that have a symmetry center.

After the development of the first laser (54), Davydov et al.(55) reported a strong second-harmonic frequency generationin organic molecules, in which electron donor and acceptorgroups are connected through a benzene ring. They concludedthat dipolarπ-conjugated molecules with electron donor andacceptor groups of opposite nature contribute to a large second-order optical nonlinearity (â) arising from intramolecular chargetransfer between donor and acceptor upon polarization. This(donor)-(delocalizedπ-conjugated connector)-(acceptor) ar-chitecture in combination with noncentrosymmetry of thechromophore is a fundamental molecular design principle fororganic NLO materials (56, 57). Recently, Uyeda et al. (57)synthesized a number of new organometallic chromophores withunusually high dynamic hyperpolarizabilities. One of thechromophores (Figure 1) combines a ruthenium(II)-terpyridinyl

Figure 1. Ruthenium(II) [5-(4′-ethynyl-(2,2′;6′,2′′-terpyridinyl))-10,-20-bis(2′,6′-bis(3,3-dimethyl-1-butyloxy)phenyl)porphinato]zinc(II)-(2,2′;6′,2′′-terpyridine)2+ (Ru-PZn). The ruthenium(II)-terpyridinylserves as a charge density acceptor, and the highly polarizableporphyrinic zinc(II) complex serves as the donor. This molecule displaysunusually large dynamic hyperpolarizabilities at frequencies relevantfor telecommunications applications (57).

P ) ø(1)‚E + ø(2):EE + ø(3) l EEE+ ... (1)

Biotechnol. Prog., 2008, Vol. 24, No. 1 75

complex as an acceptor with an ethyne-elaborated highlypolarizable porphyrinic zinc(II) donor component (Ru-PZn).

Importantly, if one is to construct an NLO material fromnoncentrosymmetric molecules with high individualâ values,the molecules cannot be ordered randomly in the bulk becausethis would render the bulkø(2) zero. Hence, noncentrosymmetricmolecules with high first hyperpolarizabilitiesâ must be orientedto exploit their optical nonlinearity. Aligning organic chro-mophores in a bulk material is typically achieved by electricfield poling, i.e., placing the molecules into a heated polymermatrix under an electric field, which polarizes and orients thechromophores (58). This presents a number of challenges, manyof which limit the maximum chromophore load per volume ofpolymer. Strong long-range electrostatic dipole interactionsbetween individual chromophore molecules (52) interfere withthe large-scale orientation of the ensemble (58). The choices ofmatrix polymers are limited, as only polymers with high glasstransition temperatures (Tg) are suitable to prevent chromophoresfrom reorienting after electric field poling and cooling of thepolymer. Chromophore migration, aggregation, and phaseseparation can occur during electric field poling or duringprocessing, The chromophore must be stable at the hightemperatures used during poling and non-traditional poling (52).On the other hand, encapsulating the chromophore into a proteinmatrix would be a first step toward designing a protein thatshields the long-range chromophores, specifically their dipole-dipole interactions, from each other, and can ultimately betailored to facilitate self-assembly into higher-order structuresthrough designed protein-protein interfaces (59, 60).

Computational Design of Four-Helix Bundle Proteinswith Nonbiological Cofactors

Computational protein design involves the modeling andidentification of protein structure and sequence, often resultingin novel structures and sequences (61-74). Such methods arenecessary for the design of systems with nonbiological cofactors,as it may not be possible to find an appropriate scaffold that

can accommodate these cofactors among natural protein struc-tures. Using such methods, Cochran et al. (63) designed a four-helix bundle protein, PAtet, that selectively binds a nonbiologicaliron diphenylporphyrin cofactor. This cofactor specificity is aresult of the computational design of interactions with the proteinthat are tailored to the nonbiological cofactor. The use ofcomputational protein design was also instrumental in identify-ing appropriate hydrophobic core packing, hydrophobic helix-helix interfaces, and complementary charge patterning to enforcean antiparallel,D2-symmetric bundle topology. SCADS, astatistical computationally assisted design strategy, was usedto identify site-specific amino acid probabilities from a givenbackbone structure (61, 62). Many successful designs in recentyears support the robustness of the method across various typesof backbone structures (62-66). The method takes as input (a)a target structure, in this case, a computationally generatedbackbone structure (75, 76), (b) the energy functions thatquantify sequence-structure compatibility, and (c) a set ofenergetic or identity constraints. Cochran et al.’s target structureincluded the locations of four fixed His residues that provideaxial ligands for the cofactor and Thr residues that determinethe local conformations of the His side chains via hydrogenbonding. The output is a set of probabilities of the amino acidsat each variable position in the protein. The resulting protein,PAtet, forms a stableR-helical homotetramer in the presence ofthe nonbiological cofactor but not in the presence of heme.

PAtet is an important first step in crafting protein systemscontaining nonbiological cofactors. In recent work (77), thiseffort has been extended to design a single-chain protein, PASC,which binds two molecules of the same cofactor (Figure 2a).PASChas several advantages compared to PAtet. Tetrameric four-helix bundles are challenging systems to work with, as theamphiphilic helices may aggregate. Single-chain proteins areeasily realized in large quantities using protein overexpressionin host organisms such asE. coli, and modification of thesequence is straightforward using site-directed mutagenesis.Stable proteins with well-packed interiors are more easily

Figure 2. Computationally designed four-helix bundle protein that binds fully synthetic nonbiological cofactors. (a) A single-chain protein, PASC,binding bis-His-ligated (hexacoordinated) iron diphenylporphyrins (77). (b) Non-symmetric hydrophobic core of the same protein-cofactor complex.Hydrophobic interior residues are rendered in yellow.

76 Biotechnol. Prog., 2008, Vol. 24, No. 1

realized since the resulting protein need not have two- or four-fold symmetry. This reduction of symmetry can also be usedto arrive at proteins that bind asymmetric cofactors. Figure 2billustrates this point, displaying the tightly packed asymmetrichydrophobic core of the single-chain variant. PASC features awell-packed hydrophobic core, complementary helix-helix andhelix-cofactor interactions, and patterned salt bridges. Impor-tantly, PASCalso exhibits experimental properties consistent witha well-folded (non-fluctuating) structure, such as well-resolved1H-15N HSQC NMR resonances.

Future work will consider the design of tetrameric four-helixbundle architectures, similar to those of Cochran et al. (63),which contain diphenylporphyrin cofactors with a zinc centerion (PZn cofactor). This has consequences for the geometry ofthe coordination site. Often zinc porphyrins are only five-pointcoordinated, and as a result future designed systems will haveone axial His ligand, while the distal site is occupied by ahydrophobic residue. Such designs will explore the requirementsof five-point coordination of the nonbiological zinc diphenylpor-phyrin in a de novo designed four-helix bundle. Finally, one ofthe prospective applications of four-helix bundle proteins is tobind nonbiological cofactors with relevance for new materials,such as the Ru-PZn cofactor (see Figure 1). Research in thisdirection is currently under way, and realizing single-chain PASC

and pentacoordinated PZn are important steps toward four-helixbundles that bind complex cofactors such as Ru-PZn.

Summary

Computational design of four-helix bundle proteins may beused to realize systems that bind nonbiological cofactors. Suchefforts are relevant for the development of new biomaterials asthey combine knowledge from the area of heme protein designwith research directions in the area of nonlinear optical (NLO)materials. The large body of knowledge from heme proteindesign, in particular that concerning the influence of thecoordinating side chains and the local cofactor environment onthe electrochemistry of the bound heme, is expected to bebeneficial for the de novo design of proteins that bind novelNLO chromophores based on porphyrins. Such proteins canprovide well-controlled local molecular environments for bind-ing and modulating recently developed NLO metalloporphyrinchromophores, which display unusually large second-orderhyperpolarizabilities. In order to exploit these superior NLOproperties in a bulk material, it is necessary to vectorially orientthe cofactors. One possible route to achieve cofactor orientationis through specifically designed protein exteriors of the four-helix bundle proteins discussed.

Acknowledgment

The authors gratefully acknowledge primary support fromthe U.S. Department of Energy (DE-FG02-04ER46156), theUniversity of Pennsylvania’s Nano/Bio Interface Center throughthe National Science Foundation (NSF) NSEC DMR-0425780.Support is also acknowledged from the National Institutes ofHealth (GM61267, GM71628), and Laboratory for Researchon the Structure of Matter through NSF MRSEC DMR05-20020. The figures were rendered using PyMol (DeLanoScientific LLC).

References and Notes

(1) Ratner, B. D.; Bryant, S. J. Biomaterials: where we have beenand where we are going.Annu. ReV. Biomed. Eng.2004, 6, 41-75.

(2) Kopecek, J. Smart and genetically engineered biomaterials and drugdelivery systems.Eur. J. Pharm. Sci.2003, 20 (1), 1-16.

(3) Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. Peptide-based stimuli-responsive biomaterials.Soft Matter2006, 2 (10), 822-835.

(4) Woolfson, D. N.; Ryadnov, M. G. Peptide-based fibrous bioma-terials: some things old, new and borrowed.Curr. Opin. Chem. Biol.2006, 10 (6), 559-567.

(5) Zhang, S. G. Fabrication of novel biomaterials through molecularself-assembly.Nat. Biotechnol.2003, 21 (10), 1171-1178.

(6) Langer, R.; Tirrell, D. A. Designing materials for biology andmedicine.Nature2004, 428 (6982), 487-492.

(7) Maskarinec, S. A.; Tirrell, D. A. Protein engineering approachesto biomaterials design.Curr. Opin. Biotechnol.2005, 16 (4), 422-426.

(8) Fairman, R.; Akerfeldt, K. S. Peptides as novel smart materials.Curr. Opin. Struct. Biol.2005, 15 (4), 453-463.

(9) Anfinsen, C. B. Principles that govern the folding of protein chains.Science1973, 181 (4096), 223-230.

(10) Izarzugaza, J. M. G.; Granja, O.; Tress, M. L.; Valencia, A.; Clarke,N. D. Assessment of intramolecular contact predictions for CASP7.Proteins: Struct., Funct., Bioinf.2007, 69 (Suppl. 8), 152-158.

(11) Vincent, J. J.; Tai, C. H.; Sathyanarayana, B. K.; Lee, B.Assessment of CASP6 predictions for new and nearly new foldtargets.Proteins: Struct., Funct., Bioinf.2005, 61 (Suppl. 7), 67-83.

(12) Wang, L.; Schultz, P. G. Expanding the genetic code.Angew.Chem., Int. Ed. 2005, 44 (1), 34-66.

(13) Budisa, N. Prolegomena to future experimental efforts on geneticcode engineering by expanding its amino acid repertoire.Angew.Chem., Int. Ed. 2004, 43 (47), 6426-6463.

(14) Link, A. J.; Mock, M. L.; Tirrell, D. A. Non-canonical aminoacids in protein engineering.Curr. Opin. Biotechnol.2003, 14 (6),603-609.

(15) Sarikaya, M.; Tamerler, C.; Jen, A. K. Y.; Schulten, K.; Baneyx,F. Molecular biomimetics: nanotechnology through biology.Nat.Mater. 2003, 2 (9), 577-585.

(16) Gibney, B. R. Heme Protein Database, http://heme.chem.colum-bia.edu/heme.php (accessed January 8, 2008).

(17) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein DataBank.Nucleic Acids Res.2000, 28 (1), 235-242.

(18) Lombardi, A.; Nastri, F.; Pavone, V. Peptide-based heme-proteinmodels.Chem. ReV. 2001, 101 (10), 3165-3189.

(19) Paoli, M.; Marles-Wright, J.; Smith, A. Structure-function rela-tionships in heme-proteins.DNA Cell Biol.2002, 21 (4), 271-280.

(20) Reedy, C. J.; Gibney, B. R. Heme protein assemblies.Chem. ReV.2004, 104 (2), 617-649.

(21) Dolphin, D.The Porphyrins; Academic Press, Inc.: New York,1978/79; Vols. 1-7.

(22) Battersby, A. R.; Leeper, F. J. Biosynthesis of the pigments oflife - Mechanistic studies on the conversion of porphobilinogen touroporphyrinogen-III.Chem. ReV. 1990, 90 (7), 1261-1274.

(23) Battersby, A. R. How nature builds the pigments of life-theconquest of vitamin B-12.Science1994, 264 (5165), 1551-1557.

(24) Battersby, A. R. Natures pathways to the pigments of life.Nat.Prod. Rep.1987, 4 (1), 77-87.

(25) Leeper, F. J. The biosynthesis of porphyrins, chlorophylls, andvitamin-B12.Nat. Prod. Rep.1989, 6 (2), 171-203.

(26) Izadi, N.; Henry, Y.; Haladjian, J.; Goldberg, M. E.; Wandersman,C.; Delepierre, M.; Lecroisey, A. Purification and characterizationof an extracellular heme-binding protein, HasA, involved in hemeiron acquisition.Biochemistry1997, 36 (23), 7050-7057.

(27) Horton, P.; Whitmarsh, J.; Cramer, W. A. On the specific site ofaction of 3-(3,4-dichlorophenyl)-1,1-dimethylurea in chloroplasts-Inhibition of a dark acid-induced decrease in midpoint potential ofcytochrome B-559.Arch. Biochem. Biophys.1976, 176 (2), 519-524.

(28) Iwata, S.; Lee, J. W.; Okada, K.; Lee, J. K.; Iwata, M.; Rasmussen,B.; Link, T. A.; Ramaswamy, S.; Jap, B. K. Complete structure ofthe 11-subunit bovine mitochondrial cytochrome bc(1) complex.Science1998, 281 (5373), 64-71.

(29) Brandt, U.; Trumpower, B. L. The protonmotive Q-cycle inmitochondria and bacteria.Crit. ReV. Biochem. Mol. Biol.1994, 29(3), 165-197.

Biotechnol. Prog., 2008, Vol. 24, No. 1 77

(30) Hunte, C.; Palsdottir, H.; Trumpower, B. L. Protonmotivepathways and mechanisms in the cytochrome bc(1) complex.FEBSLett. 2003, 545 (1), 39-46.

(31) Discher, B. M.; Koder, R. L.; Moser, C. C.; Dutton, P. L.Hydrophillic to amphiphilic design in redox protein maquettes.Curr.Opin. Chem. Biol.2003, 7 (6), 741-748.

(32) Robertson, D. E.; Farid, R. S.; Moser, C. C.; Urbauer, J. L.;Mulholland, S. E.; Pidikiti, R.; Lear, J. D.; Wand, A. J.; DeGrado,W. F.; Dutton, P. L. Design and synthesis of multi-heme proteins.Nature1994, 368 (6470), 425-431.

(33) Ho, S. P.; DeGrado, W. F. Design of a 4-helix bundle protein-Synthesis of peptides which self-associate into a helical protein.J.Am. Chem. Soc.1987, 109 (22), 6751-6758.

(34) Handel, T.; DeGrado, W. F. De novo design of a Zn-2+-bindingprotein.J. Am. Chem. Soc.1990, 112 (18), 6710-6711.

(35) Handel, T. M.; Williams, S. A.; DeGrado, W. F. Metal-iondependent modulation of the dynamics of a designed protein.Science1993, 261 (5123), 879-885.

(36) Choma, C. T.; Lear, J. D.; Nelson, M. J.; Dutton, P. L.; Robertson,D. E.; DeGrado, W. F. Design of a heme-binding 4-helix bundle.J.Am. Chem. Soc.1994, 116 (3), 856-865.

(37) Gibney, B. R.; Rabanal, F.; Reddy, K. S.; Dutton, P. L. Effect offour helix bundle topology on heme binding and redox properties.Biochemistry1998, 37 (13), 4635-4643.

(38) Gibney, B. R.; Rabanal, F.; Skalicky, J. J.; Wand, A. J.; Dutton,P. L. Iterative protein redesign.J. Am. Chem. Soc.1999, 121 (21),4952-4960.

(39) Huang, S. S.; Koder, R. L.; Lewis, M.; Wand, A. J.; Dutton, P.L. The HP-1 maquette: From an apoprotein structure to a structuredhemoprotein designed to promote redox-coupled proton exchange.Proc. Natl. Acad. Sci. U.S.A.2004, 101 (15), 5536-5541.

(40) Ghirlanda, G.; Osyczka, A.; Liu, W. X.; Antolovich, M.; Smith,K. M.; Dutton, P. L.; Wand, A. J.; DeGrado, W. F. De novo designof a D-2-symmetrical protein that reproduces the diheme four-helixbundle in cytochrome bc(1).J. Am. Chem. Soc.2004, 126 (26),8141-8147.

(41) Rau, H. K.; Haehnel, W. Design, synthesis, and properties of anovel cytochrome b model.J. Am. Chem. Soc.1998, 120(3), 468-476.

(42) Mutter, M.; Vuilleumier, S. A chemical approach to proteindesign-Template-assembled synthetic proteins (TASP).Angew.Chem., Int. Ed. Engl.1989, 28 (5), 535-554.

(43) Kamtekar, S.; Schiffer, J. M.; Xiong, H. Y.; Babik, J. M.; Hecht,M. H. Protein design by binary patterning of polar and nonpolaramino-acids.Science1993, 262 (5140), 1680-1685.

(44) Rojas, N. R. L.; Kamtekar, S.; Simons, C. T.; McLean, J. E.;Vogel, K. M.; Spiro, T. G.; Farid, R. S.; Hecht, M. H. De novoheme proteins from designed combinatorial libraries.Protein Sci.1997, 6 (12), 2512-2524.

(45) Wei, Y. N.; Liu, T.; Sazinsky, S. L.; Moffet, D. A.; Pelczer, I.;Hecht, M. H. Stably folded de novo proteins from a designedcombinatorial library.Protein Sci.2003, 12 (1), 92-102.

(46) Wei, Y.; Kim, S.; Fela, D.; Baum, J.; Hecht, M. H. Solutionstructure of a de novo protein from a designed combinatorial library.Proc. Natl. Acad. Sci. U.S.A.2003, 100 (23), 13270-13273.

(47) Marder, S. R.; Kippelen, B.; Jen, A. K. Y.; Peyghambarian, N.Design and synthesis of chromophores and polymers for electro-optic and photorefractive applications.Nature 1997, 388 (6645),845-851.

(48) Prasad, P. N.; Williams, D. J.Introduction to Nonlinear OpticalEffects in Molecules and Polymers; John Wiley & Sons: New York,1991.

(49) Sutherland, R. L.Handbook of Nonlinear Optics; Marcel Dek-ker: New York, 1996.

(50) Wolff, J. J.; Wortmann, R. Organic materials for second-ordernon-linear optics. InAdV. Phys. Org. Chem.1999, 32, 121-217.

(51) Dalton, L. Nonlinear optical polymeric materials: From chro-mophore design to commercial applications. InPolymers forPhotonics Applications I2002, 158, 1-86.

(52) Dalton, L. R.; Steier, W. H.; Robinson, B. H.; Zhang, C.; Ren,A.; Garner, S.; Chen, A. T.; Londergan, T.; Irwin, L.; Carlson, B.;Fifield, L.; Phelan, G.; Kincaid, C.; Amend, J.; Jen, A. Frommolecules to opto-chips: organic electro-optic materials.J. Mater.Chem.1999, 9 (9), 1905-1920.

(53) Verbiest, T.; Houbrechts, S.; Kauranen, M.; Clays, K.; Persoons,A. Second-order nonlinear optical materials: recent advances inchromophore design.J. Mater. Chem.1997, 7 (11), 2175-2189.

(54) Maiman, T. H. Stimulated optical radiation in ruby.Nature1960,187 (4736), 493-494.

(55) Davydov, B. L.; Derkacheva, L. D.; Dunina, V. V.; Zhabotin, M.E.; Zolin, V. F.; Koreneva, L. G.; Samokhin, M. A. Connectionbetween charge transfer and laser second harmonic generation.JETPLett.-USSR1970, 12 (1), 16.

(56) Nalwa, H. S.; Watanabe, T.; Miyata, S. Organic materials forsecond-order nonlinear optics. InNonlinear Optics of OrganicMolecules and Polymers; Nalwa, H. S., Miyata, S., Eds.; CRCPress: Boca Raton 1997; pp 89-350.

(57) Uyeda, H. T.; Zhao, Y. X.; Wostyn, K.; Asselberghs, I.; Clays,K.; Persoons, A.; Therien, M. J. Unusual frequency dispersion effectsof the nonlinear optical response in highly conjugated (polypyridyl)-metal-(porphinato)zinc(II) chromophores.J. Am. Chem. Soc.2002,124 (46), 13806-13813.

(58) Shi, Y. Q.; Zhang, C.; Zhang, H.; Bechtel, J. H.; Dalton, L. R.;Robinson, B. H.; Steier, W. H. Low (sub-1-volt) halfwave voltagepolymeric electro-optic modulators achieved by controlling chro-mophore shape.Science2000, 288 (5463), 119-122.

(59) MacPhee, C. E.; Woolfson, D. N. Engineered and designedpeptide-based fibrous biomaterials.Curr. Opin. Solid State Mater.Sci.2004, 8 (2), 141-149.

(60) Woolfson, D. N. The design of coiled-coil structures and as-semblies. InFibrous Proteins: Coiled-Coils Collagen and Elas-tomers; Academic Press: New York, 2005; Advances in ProteinChemistry Vol. 70; p 79.

(61) Kono, H.; Saven, J. G. Statistical theory for protein combinatoriallibraries. Packing interactions, backbone flexibility, and the sequencevariability of a main-chain structure.J. Mol. Biol. 2001, 306 (3),607-628.

(62) Calhoun, J. R.; Kono, H.; Lahr, S.; Wang, W.; DeGrado, W. F.;Saven, J. G. Computational design and characterization of amonomeric helical dinuclear metalloprotein.J. Mol. Biol.2003, 334(5), 1101-1115.

(63) Cochran, F. V.; Wu, S. P.; Wang, W.; Nanda, V.; Saven, J. G.;Therien, M. J.; DeGrado, W. F. Computational de novo design andcharacterization of a four-helix bundle protein that selectively bindsa nonbiological cofactor.J. Am. Chem. Soc.2005, 127 (5), 1346-1347.

(64) Swift, J.; Wehbi, W. A.; Kelly, B. D.; Stowell, X. F.; Saven, J.G.; Dmochowski, I. J. Design of functional ferritin-like proteins withhydrophobic cavities.J. Am. Chem. Soc.2006, 128 (20), 6611-6619.

(65) North, B.; Cristian, L.; Stowell, X. F.; Lear, J. D.; Saven, J. G.;DeGrado, W. F. Characterization of a membrane protein foldingmotif the ser zipper, using designed peptides.J. Mol. Biol. 2006,359 (4), 930-939.

(66) Nanda, V.; Rosenblatt, M. M.; Osyczka, A.; Kono, H.; Getahun,Z.; Dutton, P. L.; Saven, J. G.; DeGrado, W. F. De novo design ofa redox-active minimal rubredoxin mimic.J. Am. Chem. Soc.2005,127 (16), 5804-5805.

(67) Shifman, J. M.; Mayo, S. L. Modulating calmodulin bindingspecificity through computational protein design.J. Mol. Biol.2002,323 (3), 417-423.

(68) Shiamoka, M.; Shifman, J. M.; Jing, H.; Tagaki, J.; Mayo, S. L.;Springer, T. A. Computational design of an integrin I domainstabilized in the open high affinity conformation.Nat. Struct. Biol.2000, 7 (8), 674-678.

(69) Dahiyat, B. I.; Mayo, S. L. De novo protein design: Fullyautomated sequence selection.Science1997, 278 (5335), 82-87.

(70) Kuhlman, B.; Dantas, G.; Ireton, G. C.; Varani, G.; Stoddard, B.L.; Baker, D. Design of a novel globular protein fold with atomic-level accuracy.Science2003, 302 (5649), 1364-1368.

(71) Korkegian, A.; Black, M. E.; Baker, D.; Stoddard, B. L.Computational thermostabilization of an enzyme.Science2005, 308(5723), 857-860.

(72) Marvin, J. S.; Hellinga, H. W. Conversion of a maltose receptorinto a zinc biosensor by computational design.Proc. Natl. Acad.Sci. U.S.A.2001, 98 (9), 4955-4960.

(73) Looger, L. L.; Dwyer, M. A.; Smith, J. J.; Hellinga, H. W.Computational design of receptor and sensor proteins with novelfunctions.Nature2003, 423 (6936), 185-190.

78 Biotechnol. Prog., 2008, Vol. 24, No. 1

(74) Dwyer, M. A.; Looger, L. L.; Hellinga, H. W. Computationaldesign of a biologically active enzyme.Science2004, 304 (5679),1967-1971.

(75) North, B.; Summa, C. M.; Ghirlanda, G.; DeGrado, W. F. D-n-Symmetrical tertiary templates for the design of tubular proteins.J.Mol. Biol. 2001, 311 (5), 1081-1090.

(76) Betz, S. F.; DeGrado, W. F. Controlling topology and native-likebehavior of de novo-designed peptides: Design and characterizationof antiparallel four-stranded coiled coils.Biochemistry1996, 35 (21),6955-6962.

(77) Bender, G. M.; Lehmann, A.; Zou, H.; Cheng, H.; Fry, H. C.;Engel, D.; Therien, M. J.; Blasie, J. K.; Roder, H.; Saven, J. G.;DeGrado, W. F. De novo design of a single-chain diphenyl-porphyrin metalloprotein.J. Am. Chem. Soc.2007, 129(35), 10732-10740.

Received June 2, 2007. Accepted December 5, 2007

BP070178Q

Biotechnol. Prog., 2008, Vol. 24, No. 1 79