Methods in Biochemistry
Methods in Biochemistry BIOCHEM 660
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gt t J E U T E 3 8 to N 2 2 2 i e M inireview Detergents as Tools in Membrane Biochemistry Published JBC Papers in Press June 29 2001 DOI 101074jbcR100031200 R Michael Garavitoi and Shelagh FergusonMiller From the Department of Biochemistry and Molecular Biology Michigan State University East Lansing Michigan 488241319 Detergents are invaluable tools for studying mem brane proteins However these deceptively simple am phipathic molecules exhibit complex behavior when they selfassociate and interact with other molecules The phase behavior and assembled structures of deter gents are markedly in uenced not only by their unique chemical and physical properties but also by concentra tion ionic conditions and the presence of other lipids and proteins In this minireview we discuss the various aggregate forms detergents assume and some miscon ceptions about their structure The distinction between detergents and the membrane lipids that they may or may not replace is emphasized in the most recent high resolution structures of membrane proteins Detergents are clearly friends and foes but with the knowledge of how they work we can use the increasing variety of detergents to our advantage Over the past decade our understanding of the structure and function of membrane proteins has advanced signi cantly as well as how their detailed characterization can be approached experi mentally Detergents have played signi cant roles in this effort They serve as tools to isolate solubilize and manipulate mem brane proteins for subsequent biochemical and physical character ization Many of the successful methods for reconstituting 1 and crystallizing 27 4 membrane proteins rely on the unique behavior of detergents Although many new detergents are now available for use with membrane proteins their behavior in solution and in the presence of protein may limit their use with speci c experimental techniques Hence the choice of detergent and experimental con ditions will have an enormous impact on whether a technique can be successfully applied to a speci c membrane protein A clear understanding of basic detergent behavior and of the structure of micelles and protein detergent complexes is thus crucial for mem brane biochemists In this minireview we will brie y discuss the basic aspects of detergent physical chemistry that affect membrane proteins and their manipulation in the context of the new information about membrane protein structure and function The reader is directed to comprehensive reviews by Helenius and Simons 5 Tanford and Reynolds 6 Helenius et al 7 Kuhlbrandt 4 and Zulauf 8 This minireview will be reprinted in the 2001 Minireview Compendium which will be available in December 2001 This is the third article of four in the Membrane Protein Structural Biology Minireview Series Some of the work discussed in this minireview was supported in part by National lnsti tutti ogIH alth Grants P01 GM57323 to R M G and S F M and HL56773 to This minireview is dedicated to Drs Jacqueline A Reynolds and the late Martin Zulauf who gave one of us R M G invaluable insights into the behavior of detergents o whom correspondence may be addressed Tel 5173559724 Fax 5173539334 Email garavitomsuedu o whom correspondence may be addressed Tel 5173550199 Fax 5173539334 Email fergus20msuedu This paper is available on line at httpwwwjbcorg THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 276 NO 35 Issue OfAugust 31 pp 32403732406 2001 2001 by The American Society for Biochemistry and Molecular Biology Inc Printed in USA which cover the action and behavior of detergents from a biochem ical viewpoint Excellent monographs by Tanford 9 and Rosen 10 as well as a review by Wennerstr m and Lindman 11 de scribe the physical chemistry of detergents and surfactants in detail Detergents and Lipids as Surfactants Detergents are surface active molecules that self associate and bind to hydrophobic surfaces in a concentration dependent manner 8 10 11 The amphipathic character of detergents is evident in their structures Fig 1a which consist of a polar or charged head group and a hydrophobic tail Most detergents fall into one of three categories depending on the type of head group ionic cationic or anionic nonionic and zwitterionic The behavior of a speci c de tergent is dependent on the character and stereochemistry of the head group and tail In the broader sense detergents and lipids are both surfactants What distinguishes one from the other are the concentration re gimes for self association and the kinds of multimolecular struc tures each can make The problem of isolating native membrane proteins from lipid bilayers and then subsequently manipulating them is in essence a problem of dealing with mixed surfactant systems The most common question about detergent use is whether a magic bullet detergent exists The simple answer is no but successful strategies for detergent use do exist The key to a successful experiment is to understand how detergents and lipids impact the physical nature of a protein detergent lipid complex and its behavior The Micelle What Is It Detergent monomers in aqueous solutions are involved in two kinds of basic phase transitions First monomers can crystallize in aqueous solution 10 although the majority of detergents used in membrane biochemistry do not 477 Second detergent monomers self associate to form structures called micelles 8 10 11 At a broad threshold of monomer concentration called the critical mi celle concentration CMC1 Fig 1b self association occurs and micelles form Ideally the concentration of detergent monomers stays constant above the CMC as more detergent is added to the solution only the concentration of micelles increases 12 When the concentration exceeds the CMC a detergent becomes capable of solubilizing hydrophobic and amphipathic molecules such as lip ids into mixed micelles or micellar aggregates 10 Moreover the complete and stable solubilization of many integral membrane proteins generally occurs above the CMC as the detergent associ ates with the hydrophobic surfaces of membrane proteins to create water soluble protein detergent complexes PDCs 13715 Micellarization is a common phenomenon with many surfac tants The average size and shape of micelles s 3439 E U E 396 2 2 M 2 H g 2 b e 32404 H a Ma a OG OO DOOH C8E5 CH3 NCH3 LDAO O b I I I 2 10 Cam 5 monomers g a 5 39 a micelles 0 El l 0 5 10 15 SDSt0ml mM FIG 1 Detergent structure and micellarization Detergent mono mers of BOG octylpentaoxyethylene C8E5 and lauryl dimethylamine oxide LDAO are shown in a each consists of a polar head group and N alkyl tail In b the change in concentration SDStht of monomer and micellar fractions versus the total detergent concentration is shown for SDS The CMC is the threshold detergent concentration where micelles begin to form However the CMC is not truly a sharp boundary as the physical changes being followed light scattering surface tension etc show broad transitions around the CMC dashed lines Thus the CMC is often the midpoint of a concentration range dotted lines The gure shown in b was adapted from Ref 12 reprinted with permission copyright 1980 American Chemical Society H FIG 2 Space filling models of BDoctyl glucoside micelles classical I in a 0quot micelle in b and 50monomer mi celle in c The micelles shown in b and c were derived from 40 ns molecular dynamics simulation data 17 and have nonspherical and nonuniform shapes The polar portions of the detergents oxygen atoms are red carbon atoms gray do not cover completely the micelle surface Hence substantial portions of the core are exposed to bulk solvent including alkyl chains lying along the micelle surface arrowheads Fig 2 b and c have the hydrophobic tails packing in a much more disorganized but compact fashion 17 18 TWO consequences of micelle structure are now clearly evident 1 the micelle surface is quite rough and heterogeneous in character and 2 not all hydro phobic tails are buried or point toward the center of the micelle Hence micelle radii are about 10 30 smaller than the fully extended length of the detergent monomer 8 and many of the hydrophobic tails have considerable contact with water and sol utes Moreover molecular dynamics studies 17 18 also show that micelle shape is very dependent on aggregation number Fig 2 b and c and that the concept of a spherical micelle really denotes only an average shape The concept of a compact disordered micelle clearly suggests that monomer packing defects could radically affect the size shape and behavior of micelles As lipids other detergents or amphipathic solutes are incorporated into the micelles of a pure detergent to form mixed micelles packing defects may be introduced or on the other hand eliminated By extrapolation the bound detergents in a PDC are unlikely to be well ordered and ef ciently packed Perhaps the inability of certain detergents to solubilize or stabilize some mem brane proteins arises from the unstable defectridden packing of detergent monomers on the surface of the protein Minireview Detergents in Membrane Biochemistry Another misconception is that micelles are static structures of uniform shape The term monodisperse is often applied to colloidal systems to signify a uniform size and shape of a population of parti cles For detergents monodispersity is better perceived to be a lack of detectable heterogeneity in the average micelle size and shape 19 The experimental evidence suggests that micelles are quite uid and rapidly exchange micellar components with the solvent 10 11 20 21 Micelles of small detergents can exhibit dramatic uctuations in micellar shape they can deform split and fuse over time 10 11 17 18 For some detergents appreciable changes in micelle aggregation number size and shape may occur as the total detergent concentra tion rises 22 23 Changes in micelle shape from spherical to ellip soidal or even rodlike occur with many pure detergents 22 23 but may be even more common when a detergent is mixed with another detergent lipid or protein 24 Surfactant Phase Behavior Selfassociation and crystallization are only two of many possible phase transitions that surfactant solutions may exhibit 8 10 Phase diagrams of detergent behavior in aqueous solutions are generally simple for the nonionic detergents with N alkyl tails of 8 carbons Fig 3 Nonionic and zwitterionic detergents with N alkyl tails of 12 carbons or longer tend to exhibit much more complex phase behaviors Fig 3 where some phase changes involve micel lar growth andor fusion to form mesophases with distinct struc tural properties 8 10 16 One common detergent phenomenon is called the cloud point 8 16 where a clear homogeneous detergent solution turns turbid upon heating The formerly single liquid phase L1 eventually separates into two immiscible solutions Ll L1 one detergentrich and the other detergentpoor The boundary between the isotropic detergent phase and the coexist ence of the two liquid phases Fig 3 is called a consolute boundary 8 16 Bordier 25 recognized that this phase phenomenon could be exploited for membrane protein purification and the technique of detergent phase separation is still used today 26 The phase transitions exhibited by a particular surfactant are determined by its monomer structure shape as well as its chem istry 8 16 eg its ionization state or capacity for hydration Thus changes in the solvent environment can also alter the nature of surfactant aggregation 8 27 The mere addition of salts or polar solutes to a detergent solution can radically alter the phase behav ior of a detergent system causing phases to appear well below the relatively high detergent concentrations seen with the pure deter gents 8 16 The cloud point phase separation is a frequent prob lem during membrane protein crystallization 2 4 and is easily induced by a number of variables eg detergent type salt tem perature and precipitant For example the octyloligooxyethylene CgEm detergents display a lower consolute LC boundary Fig 3 As the temperature rises micelles aggregate into clusters 8 23 until these clusters phase out to form a new aqueous detergent rich phase The addition of salt also depresses the LC boundary to lower temperatures 8 27 In contrast the addition of polyethyl ene glycol to solutions of alkyl glycoside detergents such as BD octyl glucoside BOG and BDdecyl maltoside causes an upper consolute UC boundary to appear Fig 3 The take home lesson is that solution and environmental parameters affect not only the basic detergent phenomenon we rely on micellarization but also whether other detergent phases appear or not Mixed Micelles ProteinDetergent Complexes and Crystallization What makes understanding surfactant phase phenomena so im portant to membrane biochemists is that the mere use of deter gents with membrane proteins forces us to confront them from protein isolation to crystallization to reconstitution How a mem brane protein behaves will be in uenced by detergentprotein and detergentdetergent interactions as well as interactions with any remaining lipid Considering only detergents and lipids it is known that mixed systems will not behave like solutions of the pure components 10 11 Hence changes in micelle shape and size CMC and phase behavior can all occur and they are not easily predicted even for simple solutions containing two detergents The addition of a membrane protein to the mix further compli cates matters The uidity and packing ef ciency of the detergent 8003 LL Jeqweldes uo uosgpewugsuoong lo Augienun 1e 6J039OqI39MMM U401 pepeolumoa 2 g U E 3 u 2 2 on V o 8 3 Ir fie Minireview Detergents in Membrane Biochemistry 32405 CSES C12E8 BOG 100 100 100 rut Lquot L L LI L UC T C 5 LC L L13 nltH I L Lquot Li 0 0 o o MW 100 o o Wm 100 0 a wlw 10 FIG 3T r sus L a L base I a 4 LII CSE5 dodecyloctaoxyethylene C RES L ver L L p for L L a a and BOG Although the phase diagram for CgE5 is quite simple the equivalent diagram for CHE8 shows several additional phases see Refs 8 and 16 for details For CgE5 and C12E8 detergent phase separation is often seen under experimental conditions because salts and polymers may depress the LC boundary to below room temperature CHE8 also exhibits the hexagonal H1 phase hexagonal packing of rodlike micelles at 50 ww mixture with water at 30 C At a threshold detergent concentration bicontinuous cubic V 1 and lamellar L0 phases are seen 8 16 For BOG in water only the lamellar L0 and gel L 3 phases are observed aside from solid detergent S However the addition of polyethylene glycol causes the appearance of an C boundary which rises with increasing polymer or salt concentration 2 8 The phase diagrams were reproduced from Ref 8 with permission of CRC Press Inc monomers bound to the protein will affect the behavior and stabil ity of the detergent layer This may result in poor protein solubility and protein inactivationaggregation Thus detergent behavior during and after protein extraction from a bilayer will impact the isolation 13 14 28 characterization 13 15 29 and stability 13 30 of membrane proteins When considering the added effects of other solvent components salt pH etc seemingly small changes in experimental conditions may give rise to detergent effects not expected from the pure detergent How detergent behavior impacts the solubility stability and structure of PDCs is then important to know For membrane pro tein crystallization an early major emphasis was placed on creat ing simple lipidfree PDCs 3 4 using nonionic detergents that produced small almost spherical micelles 8 31 to control the shape size and behavior of the PDC It was soon recognized that detergentdependent phase transitions had an enormous impact on crystallization Unwanted phase behavior could prevent crystal growth 32 and even denature protein 33 However in many cases crystal growth often occurred as conditions approached an upper or lower consolute phase boundary 3 Since then much effort has focused on understanding the relationship between detergentdependent phase behavior of the PDC and crystal growth 15 29 as well as how the characteristics of the PDC can be altered by different detergents 2 3 31 32 and the addition of small amphiphilic consolutes 15 34 35 The characterization of membrane protein crystals by single crystal neutron diffraction and D20H20 density matching 36 39 has provided a wealth of information about the shape and structure of a PDC For example the structures of OmpF porin from Esche richia coli in different detergents and crystal forms revealed some interesting aspects about detergent behavior PebayPeyroula et al 36 studied the tetragonal crystal form of OmpF porin containing decyldimethylamineoxide or BOG With decyldimethylamine oxide the PDC behaved as a hard surface complex see Fig 2 in PebayPeyroula et al 36 where the detergent layer appeared as a discrete and continuous torus about the protein In contrast the porinBOG complex revealed a partial fusion of the detergent torus with its neighbors see Fig 6 in PebayPeyroula et al 36 When Penel et al 37 looked at the trigonal crystal form of OmpF porin containing octylhydroxyethylsulfoxide see Fig 4 in Penel et al 37 the detergent torus about each porin molecule had completely fused with its nearest neighbors to create a continuous detergent domain within the crystal Clearly detergents that should nor mally just produce small spherical or ellipsoidal micelles can be induced to form more complex structures at concentrations below 50 ww Moreover detergentdetergent interactions are often an integral part of the long range structure in membrane protein crystals If detergent interactions and structure play a role in membrane protein crystal growth and integrity could more lipidlike surfac tants serve the same role Landau and Rosenbusch proposed this question and came up with a novel way of crystallizing membrane proteins 40 41 In essence a preformed surfactant phase with a more membranelike structure might be used to partition mem brane proteins into an environment that would favor close interac tions suitable for nucleating and sustaining crystal growth The bicontinuous cubic surfactant phases made by monoacyl glycerols 16 42 seem ideal for this purpose as continuous regions of solvent and surfactant extend throughout the phase and can coexist with a bulk solvent phase Detergentsolubilized membrane protein added externally can easily partition into the bicontinuous cubic phase the solvent channels allowed the manipulation of the aque ous environment to initiate crystallization Although many of the assumptions made by Landau and Rosenbusch are not con rmed their technique allowed the high resolution structure determina tion of bacteriorhodopsin 43 44 and halorhodopsin 45 Lipid Interactions as Observed in Membrane Protein Crystals The crystal structure of bacteriorhodopsin obtained from the cubic phase system discussed above 43 44 showed a remarkable feature a layer of lipid molecules was resolved on the protein surface The nature of the lipids originating from the native bac terial membrane and their positioning in the grooves and crevices of the protein Fig 4 suggest speci c and well de ned proteinlipid interactions Over the years numerous studies have demonstrated that membrane lipids are rapidly exchanging at the surface of integral membrane proteins 46 even though a motionally re stricted population was observed and quanti ed by EPR 47 The functional signi cance of this annular layer of lipid has been much debated but for many purposes the bilayer has been usefully considered as a hydrophobic solvent albeit complex in its proper ties 48 see also the rst minireview in this series by White et al 64 With the advent of high resolution crystal structures of mem brane proteins the observation of proteinbound lipid molecules now appears to be becoming a rule rather than an exception Moreover these crystalline complexes of membrane proteins and lipid do not contain just unusual lipids such as cardiolipin 49 or diether lipids 44 but also more common phospholipids The struc ture of bovine cytochrome c oxidase at 28A resolution revealed 5 phosphatidylethanolamine and 3 phosphatidylglycerol molecules per 200 kDa monomer 50 At higher resolution 20 A 14 phos pholipids including 5 cardiolipin molecules have been identi ed2 which are still only a subset of the 56 lipids with restricted mobility that have been identi ed by EPR 47 These recent crystallographic results imply that lipid may help membrane proteins assume more stable and homogeneous confor mations Hence many detergents may work best along with reten tion of some native lipid 51 In contrast complete lipid removal demands that a detergent must be able to substitute successfully for most if not all bound lipid eg dodecyl phosphocholine used in NMR structure determination 52 53 Nonetheless the mainte nance of some lipidprotein interactions may be critical for proce dures like crystallization The crystal structures of rhodopsin 54 and the sarcoplasmic Ca2 pump 55 emphasize this point In the case of rhodopsin minimal puri cation was used including a sin gle detergent extraction step 56 whereas the crystal A t 2 E U T E Di 53 3 2 2 I yrs 32406 FIG 4 A view of membrane protein interactions with lipids Native lipids are seen bound to the surface ofbacteriorhodopsin in the 155A crystal structure 44 and suggest intimate and speci c interactions between the protein and lipids to minimize s lf i tin into39 1 H p J 139 p be aggregates 28 Which is often promoted by phospholipid However complete removal of bound lipid from many membrane proteins is rarely achieved and is often detrimental to structure and function 13 57 58 Even When reasonably active forms can be maintained in detergent the structural exibilityintegrity of membrane proteins may be in uenced by the loss of associated lipid For bacteriorho dopsin NMR studies 59 clearly showed changes as native lipid was removed Finally conditions and detergents that can maintain native like activity 60 61 may still induce subtle changes that are not detectable in routine assays 57 62 63 Hence complete delipidation may not be the appropriate goal When designing pu ri cation procedures With the aim of structure determination 28 Conclusions The critical role of detergents in all aspects of membrane protein biochemistry cannot be fully addressed in the context of this short review As noted above the behavior of detergents clearly impacts membrane protein puri cation and crystallization as well as re constitution 1 which was not discussed However a few general ities can be made that apply to all systems The nature of the solubilization detergent is an important factor in determining the size and properties of the resulting PDCs Moreover the starting lipid content in the puri ed protein is a critical but often uncontrolled variable Thus we come to a new paradigm Where purer is not better and isolation of speci c proteinlipid complexes may be the more desirable goal for structural and inctional studies of mem brane proteins Banerjee et al 51 showed that different detergents extracted different kinds and amounts of lipids from the same mem brane along With protein often With signi cant differences in activ ity of the isolated protein Such care il studies may be de rigueur for the success il structural analysis of many membrane proteins AcknowledgmentsiWe thank Drs S Bogusz R M Venable and R W Pastor for allowing us access to their molecular dynamics data on the BDoctyl glucoside micelles We also thank Dr S Yoshikawa for permission to discuss unpublished observations REFERENCES 1 Rigaud J L Pitard B and Levy D 1995 Biochim Biophys Acta 1231 2237246 Garavito R M MarkovicHousley Z and Jenkins J A 1986 J Crystal Growth 76 7017709 Garavito R M Picot D and Loll P J 1995 J Bioenerg Biomembr 28 13727 K hlbrandt W 1988 Q Rev Biophys 21 4297477 Helenius A and Simons K 1975 Biochim Biophys Acta 415 69779 Tanford C and Reynolds J A 1976 Biochim Biophys Acta 457 1337170 Helenius A McCaslin D R Fries E and Tanford C 1979 Methods Enzymol 56 7347749 Zulauf M 1991 in Crystallization ofMembrane Proteins Michel H ed pp 54771 CRC Press lnc Boca Raton FL Tanford C 1980 The Hydrophobic Effect John Wiley amp Sons lnc New York Rosen M J 1978 Surfactants and Interfacial Phenomena John Wiley amp ons lnc New York Wennerstro39m H and Lindman B 1979 Phys Reports 52 1786 12 Gunnarsson G Jo39nsson B and Wennerstro39m H 1980 J Phys Chem 84 K 019 93 0099 Minireview Detergents in Membrane Biochemistry 311473121 13 Haneskog L Andersson L Brekkan E Englund A K Kameyama K Liljas L Greijer E Fischbarg J and Lundahl P 1996 Biochim Bio phys Acta 1282 39747 14 le Maire M Kwee 8 Andersen J and Muller J 1983 Eur J Biochem 129 5257532 15 Marone P A Thiyagarajan P Wagner A M and Tiede D M 1999 J Crystal Growth 207 2147225 16 Mitchell D J Tiddy G J T Waring L Bostock T and McDonald M P 1983 J Chem Soc Faraday Trans 79 97571000 17 Bogusz S Venable R M and Pastor R W 2000 J Phys Chem B 104 546275470 18 Tieleman D P van der Spoel D and Berendsen H J C 2000 J Phys Chem B 104 638076388 19 Menger F M 1979Acc Chem Res 12 1117117 20 Thomas M J Pang K Chen Q Lyles D Hantgan R and Waite M 1999 Biochim Biophys Acta 1417 1447156 21 Zhou C and Roberts M F 1997 Biochim Biophys Acta 1348 2737286 22 Nilsson PG Wennerstro39m H and Lindman B 1983 J Phys Chem 87 137771385 23 Zulauf M and Rosenbusch J P 1983 J Phys Chem 87 8567862 24 Lambert 0 Levy D Ranck J L Leblanc G and Rigaud L 1998 Biophys J 74 9187930 25 Bordier C 1981 J Biol Chem 256 160471609 26 Sivars U and Tjerneld F 2000 Biochim Biophys Acta 1474 1337146 27 Weckstrom K 1985 FEBS Lett 192 2207224 28 KraghHansen U le Maire M and Moller J V 1998 Biophys J 75 293272946 29 Hitscherich C Kaplan J Allaman M Wiencek J and Loll P J 2000 Protein Sci 9 155971566 30 De Grip W J 1982 Methods Enzymol 81 2567265 31 Timmins P A Leonhard M Weltzien H U Wacker T and Welte W 1988 FEBS ett 238 3617368 32 Garavito R M and Rosenbusch J P 1986Methods Enzymol 125 3097328 33 Michel H 1982 EMBO J 1 126771271 34 Thiyagarajan P and Tiede D M 1994 J Phys Chem 98 10343710351 35 Timmins P A Hauk J Wacker T and Welte W 1991 FEBS Lett 280 1157120 36 PebayPeyroula E Garavito R M Rosenbusch J P Zulauf M and Timmins P A 1995 Structure 3 105171059 37 Penel S Pebay Peyroula E Rosenbusch J Rurnrnel G Schirmer T and Timmins P A 1998 Biochimie Paris 80 5437551 38 Roth M Arnoux B Ducruix A and ReissHusson F 1991 Biochemistry 30 940379413 Roth M LeWittBentley A Michel H Deisenhofer J Huber R and Oesterhelt D 1989 Nature 340 6597662 40 Landau E M and Rosenbusch J P 1996Proc Natl Acad Sci U S A 93 14532714535 41 Nollert P Royant A Pebay Peyroula E and Landau E M 1999 FEBS Lett 457 2057208 Briggs J Chung H and Caffrey M 1996 J Phys II France 6 7237751 Belrhali H Nollert P Royant A Menzel C Rosenbusch J P Landau E M and Pebay Peyroula E 1999 Structure 7 9097917 44 Luecke H Schobert B Richter H T Cartailler J P and Lanyi J K 1999 J Mol Biol 291 8997911 45 Kolbe M Besir H Essen L 0 and Oesterhelt D 2000 Science 288 Review TRENDS in Microbiology Vol14 No3 March 2006 Full text provided by wwwsciencedirectcom SCIENCEltdDIRECT39 Phage display in the study of infectious diseases Lisa M Mullen Sean P Nair John M Wardz Andrew N Rycrofts and Brian Henderson1 1Division of Microbial Diseases UCL Eastman Dental Institute University College London 256 Gray s Inn Road London UK WC1X 8LD 2Department of Biochemistry and Molecular Biology University College London Gower Street London UK WC1E GBT 3Department of Pathology amp Infectious Diseases Royal Veterinary College Hawkshead Lane North Mymms Hertfordshire UK AL9 7TA Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection Phage display is a simple functional genomic methodology for screening and identifying protein ligand interactions and is widely used in epitope mapping antibody engineering and screening for receptor agonists or antagonists Phage display is also used widely in various forms including the use of fragment libraries of whole microbial genomes to identify peptide ligand and protein ligand interactions that are of importance in infection In particular this technique has proved successful in identifying microbial adhesins that are vital for colonization Phage display technology Display on filamentous phage One of the rst genetic techniques developed to study protein ligand interactions was phage display described by Smith in 1985 1 This method displays recombinant peptides or proteins on the surface of phage particles which can then be selected for panned for Figure 1 by enabling the phage to interact with selected immobilized ligands The power of phage display lies in its ability to i maintain a physical link between the displayed protein and the DNA sequence encoding it and ii screen libraries containing billions of unique peptides and proteins Escherichia coli lamentous phage of the Ff class including strains M13 fd and f1 have been extensively used to develop and exploit this technology These phage are composed of a circular singlestranded DNA genome that is encased in a long tube composed of thousands of copies of a single major coat protein with four additional minor capsid proteins at the tips Figure 2 Phage display involves the fusion of foreign DNA sequences to the phage genome such that the resulting foreign proteins are expressed in fusion with one of the coat proteins Although all ve coat proteins have been used to display proteins or peptides gene VIII protein Corresponding author Mullen LM lmulleneastmanuclacuk Available online 7 February 2006 lell and geneIIIencoded adsorption protein pIII are by far the most commonly used 2 A viable wildtype phage expresses 2700 copies of pVIII and 3 5 copies of p111 Figure 2 3 although this does depend on the size of the phage genome Phage display libraries can be constructed using vectors based on the natural Ff phage sequence ie phage vectors or by using phagemids which are hybrids of phage and plasmid vectors 23 Such phagemids are designed with the origin of replication ori from the Ff phage a plasmid origin of replication from E coli gene 111 andor gene VIII for fusion formation a multiple cloning site and an antibiotic resistance gene 2 However they lack all other phage genes that encode the structural and nonstructural proteins that are required to produce a complete phage Phagemids can be grown as plasmids in E coli and packaged as recombinant Ff phage DNA with the aid of helper phage which provide all of the necessary components for phage assembly Filamentous phage versus alternative systems for phage display The key feature of lamentous phage as applied to phage display is that in contrast to the lytic bacteriophages lamentous phage are assembled in the cytoplasmic membrane and secreted from infected bacteria without cell lysis 2 Figure 3 However the characteristics of the lamentous phage life cycle has limitations for the display of proteins the properties of which prevent the correct transfer of the hybrid capsid protein across the lipid bilayer of the inner membrane of E coli 4 Alternative bacteriophage display systems have been developed using bacteriophage such as T4 T7 4 and P4 5 which have lytic life cycles so that the proteins of the phage capsids are assembled and folded in the cytoplasm rather than being secreted through the membrane 4 Display of proteins encoded by cDNA fragments on phage Because the most common approaches to phage display described earlier involve Nterminal fusion to the gene III or gene VIII products of lamentous phage they are unsuitable for surface expression of proteins coded by wwwsciencedirectcom 0966842X see front matter 2006 Elsevier Ltd All rights reserved doi101016jtim200601006 142 TRENDS in Microbiology Vol14 No3 March 2006 iv Pan ennched phage he Ampli ed phage 7Mi 71VMlt iii Elute bound phage and amplify in E cali i Incubate phage library with immobilized ligand ii Wash off unbound phage TRENDS lli Microbiology Figure1 i intact cDNA inserts that have stop codons 67 Hence most phage libraries of cDNA fragments are constructed in alternative display systems However a modi ed lamentous phage display system based on the high a tnity interactions between the Jun and Fos leucine zipper proteins was developed by Crameri and Suter 6 In this system the Jun leucine zipper protein is fused to the N terminus of the pIII coat protein so that the Jun protein is displayed on the surface of the phage and the cDNA libraries are cloned as a Cterminal fusion to the Fos leucine zipper protein 8 The pIII anchored Jun protein is bound by the soluble Fos fusion in the periplasm thus the cDNAencoded protein is bound indirectly to pIII Natural peptide libraries versus random peptide libraries There are two types of phage display library random peptide libraries RPLs and natural peptide libraries NPLs The repertoire of peptides displayed in RPLs is encoded by synthetic random degenerate oligonucleotide inserts 9 11 and these libraries have been extensively used to identify linear antigenic epitopes see later The advantage of this type of library is their universal nature which enables many applications of each library The main iiwiu disadvantage of RPLs is that because of the way in which they are constructed peptide sequences that are not found within the antigen or intact pathogen can be displayed 12 By contrast NPLs are constructed from randomly fragmented DNA from the genomes of selected organisms such as pathogenic microbes Thus the phage particles in these libraries display fragments of natural proteins Although the majority of clones in NPLs are nonfunctional only one in 18 clones will be correctly in frame with the vector sequences one clone in three will start correctly one clone in three will end correctly and one clone in two will be in the correct orientation peptides or proteins selected from NPLs are more successful in eliciting an antibody response that crossreacts with the native intact pathogen than those selected from RPLs 13 Therefore NPLs provide important alternatives to RPLs for appli cations such as the identi cation of vaccine components and recently have been used eifectively in the identi cation of bacterial adhesins see later Phage display and infectious diseases Phage display technology has been used in a wide variety of applications including the identi cation of peptide agonists and antagonists for receptors 14 the pVII 5 pVIII 2700 W W N5 pill 5 65 nm gll gX gV ngl gll ng ngll glll ng gl glV plX 5F H 930nm TRENDS lli Microbiology Figure 2 Dimensions and architecture offiiarnentous bacteriophage fd The copy number of each protein is shown in brackets www5ciencedirectcom TRENDS in Microbiology Vol14 No3 March 2006 143 Secretion 4 Phage coat proteins 3 i ppVpV a 6 PVHL PIX 3 2 g i a Replication of 3 g g x phage genome E E 34 is E E i3 E 5 Phageencoded 5 8 E proteins i 2 T L Transcription gt Translation 7 TRENDS in Microbiology Fianna w i 39 h quot The drawingis schematic and not to scaie1 Phage oindtothe E coiiceiithroughthe piii coatprotein The singierstranded virai genome strand singie circie is injected into the ceii and a comoiementary strand at i 4 4 W aiiten V g g piX proteinsfor reoiication oii ov strand is synthesized to form a doublerstranded phage synthesis inciuding coat proteins piii pVi pVii pViii and 4 in 3 Th h 39 39 39 tne 4 39 V at 4 i and the rstrand as a temoiate 4 Virions are assemoied and exported across the oacteriai membranes identi cation of targets for the inhibition of tumour speci c angiogenesis 15 the identi cation of peptide drug candidates 16 vaccine development 17 and the isolation and engineering of recombinant antibodies 18 However in recent years numerous studies have used phage display to address speci c aspects of infectious disease The purpose of this review is to focus on the growing number of ways in which phage display technol ogy can be applied to the study of infectious diseases and to evaluate the impact of the use of this technology Investigation of host pathogen interactions Infectious diseases are absolutely dependent upon host pathogen interactions the extent of which determines the infectious process both for the pathogen and the host As discussed later epitope mapping of infectious agents has been use numerous research groups particularly to focus on the bacterial adhesins that enable host colonization Phage display technology has been used to good effect in malaria research to investigate host pathogen inter actions in both hosts of the malaria parasite Homo sapiens and the mosquito Anopheles Panning of a Plasmodium falciparum the causative agent of malaria cDNA phage display library against immobilized human erythrocyte membrane proteins identi ed seven parasite proteins that are potentially involved in the entry into or exit from human erythrocytes by P falciparum 19 Such proteins could become vaccine targets The search for the mechanisms used by Plasmodium species to cause infection has also prompted novel uses for phage display libraries The Plasmodium parasite wwwsciencedirectcom completes its life cycle in the mosquito and it is this interaction rather than that of the human host that has been investigated by Ghosh et al 20 Because the development of the Plasmodium parasite in the mosquito involves the crossing of both midgut and salivary gland epithelia this study investigated the hypothesis that such traf cking requires speci c host pathogen interaction A phage display library of random dodecapeptides fused to the N terminus of phage coat protein VIII was injected into Anopheles followed by dissection of the organs of interest and elution of bound phage A 12residue peptide from the eluted phage was identi ed and designated salivary gland and midgut peptide 1 SM1 20 This peptide strongly inhibited Plasmodium invasion of salivarygland and midgut epithelia thereby hindering the development of the parasite in Anopheles This study illustrates another variation of phage display technology the use of phage display libraries to investigate speci c phagebinding targets within the whole organism In vivo panning was rst described by Pasqualini and Ruoslahti 21 who isolated phagedisplayed peptides that bound to vascular beds in vivo To date there are limited reports of such in vivo panning but this could have an important future role for the identi cation of speci c tissue receptors for microbes or their products A variation on this theme is the panning of phage display libraries against ex vivo biomaterials that have been used in a range of clinical applications including prosthetic heart valves and intravenous catheters Bac teria such as Staphylococcus aureus and Staphylococcus epidermidis are frequently isolated from biomaterial infections 22 A phage display library constructed from the genomic DNA of S aureus was panned against a central intravenous catheter that had been removed from a patient 23 Numerous clones from the S aureus library were recovered through panning and after the second and third rounds the enriched phage encoded fragments of bacterial proteins known to bind to brinogen and BZglycoprotein I which unsurprisingly were the most abundant host proteins deposited on the catheter Lamininbinding motifs in the surface virulence factor plasminogen activator PLA of Yersinia pestis were mapped by enedek et al 24 using an RPL This approach involved identi cation of phagedisplayed amino acid sequences that bound to laminin then comparison of these sequences to the sequence of PLA to identify the speci c binding site of PLA to laminin This study demonstrates how phage display can be used to map speci c binding sites within a protein of interest Epitope mapping and identification of potential vaccine candidate antigens The term epitope can be used to describe the contacting points of any molecular interaction but it is more often used to describe the region on an antigen that elicits an immune response The identi cation of epitopes from microbial pathogens has obvious importance in the study of infectious disease particularly for the development of novel vaccines Phage display technology is especially suited to epitope mapping an has been widely used in infectiousdisease research for review see Ref 17 Much of the work on epitope mapping and identi cation of mimotopes of infectious agents has used RPLs Mimotopes are peptide sequences that are capable of inducing immune responses by mimicking the structural features of a nonlinear protein epitope or of a nonprotein eg carbohydrate epitope structure These libraries have been panned against immune sera to identify disease speci c epitopes Selected examples of these applications are listed in Table 1 In addition phage display has successfully identi ed peptide mimics of polysaccharides This approach has important implications for the develop ment of novel vaccines against encapsulated bacteria because the polysaccharide capsules of these bacteria are often antigenic Peptide mimics of the capsular TRENDS in Microbiology Vol14 No3 March 2006 polysaccharide of three of the Neisseria meningitidis serotypes A 25 B 26 and C 27 have been identi ed using RPLs The results of these studies are encouraging in that the mimotopes identi ed in this way did elicit immune responses in murine models and could be useful in the development of novel vaccines against this bacterium The pathogenesis of S aureus has been investigated by panning an RPL against the RNAIIIactivating protein RAP which autoinduces toxin production in this bacterium 2829 Initial work demonstrated that selected peptides could attenuate infection by S aureus in murine models 28 More recent studies have identi ed a peptide mimic for the RAP protein that when displayed on E coli and used to vaccinate mice prevented mortality caused by S aureus infection 29 Infection with seudomonas aeruginosa is a major problem in patients with cystic brosis CF The development of vaccines against this organism must take into account that antibiotic treatment of chronic P aeruginosa infections rarely results in clearance of the bacterium This is in contrast to treatment of early P aeruginosa infections in which the bacteria can be eradicated 30 Antigens expressed early in P aeruginosa infections of CF patients were investigated by panning an RPL against sera from noninfected and infected patients 31 In conjunction with genearray data and bioinfor matic analysis this study identi ed several genes that encode secreted proteins and outermembrane proteins that are potential targets for vaccine development against P aeruginosa infection 31 Alterations in the panning technique used such as subjecting the library to initial negative selection further increases the potential uses of these libraries Gnanasekar et al 32 constructed a T7 phage display library of the cDNA of the parasite Brugia malayi a causative agent of lymphatic lariasis To ensure that the clones of interest ie those binding to infectionspeci c antibodies were enriched the phage library was rst subjected to three negativeselection steps using noninfected human sera followed by a positiveselection step using sera from patients infected with B malayi Further experiments revealed that one of the ve antigens identi ed using this strategy B malayi Table 1 Surface display of antigenic epitopes and mimotopes on filamentous phage Library creened against Results Refs 12mer RPL displayed on plll Polyclonal sera specific for Burkholderia Identification of linear and discontinuous protease 49 pseudomallei protea epitopes of B pseudomallei 7mer RPLs displayed on plll Purified anti Mycoplasma IgG 394 quot39 39 of M antigens 50 Phage clones identified by screening induced immune responses in murine model 12mer RPL displayed on plll Monoclonal IgAI specific forthe capsule of Identification of mimotopes of the capsular poly 51 Streptococcus pneumoniae saccharide of type 8 S pneumoniae When conjugated to tetanus toxoid mimotopes induced a type 8 capsular polysaccharide specific antibody nse in mice 7mer RPL displayed on plll Sera from swine infected with Nipah virus Identification of several putative epitopes within 52 the nucleocapsid protein of Nipah virus 12mer RPL displayed on plll Polyclonal lgG specificfor neutral polysaccharides Isolation of phage clones that were antigenic 53 of Mycoplasma tuberculosis mimotopes of B cell epitopes of M tuberculosis sugars One clone invoked immune responses in rabbits www5ciencedirectcom w NIPSlike protein conferred protection against challenge infections in animal models 32 Single chain variable fragment scFv libraries have been used in several ways to study infectious diseases Identi cation of speci c scF vs that bind to microbial antigens forms the basis for the development of novel vaccines and diagnostic reagents 3 Panning of a scFv library against puri ed severe acute respiratory syn dromeassociated coronavirus SARSCoV identi ed two antibody fragments that bind to SARSCoV with high speci city 33 These could potentially be used in the development of detection assays for the virus or to elucidate potential vaccine candidate antigens Phage display scFv libraries have also been used successfully to develop immunodiagnostic and detection methods for microbial products or spores One of the most impressive aspects of these applications is the high speci city of selected clones for the microbe For example panning of a scFv library against spores of Bacillus subtilis resulted in the selection of clones that bound to only one of 11 spore strains 34 Similarly clones selected from a phage display scFv library screened against Clostridium di icile toxin B were highly speci c and showed no cross reactivity with strains of C di icile that are toxinB negative The singlechain antibody could also be used to detect as little as 10 ng of toxin B when used in ELISAs 35 Identification of bacterial adhesins Microbial infections are initiated by molecular inter actions between the pathogen through cell surface adhesins and receptor molecules on host cells and the extracellular matrix ECM resulting in microbial adhesion which can be followed by internalization 3637 It is well established that the adhesion of enteric oral and respiratory bacteria is required for colonization 37 Furthermore when bacteria adhere to surfaces they are substantially more resistant to host antimicrobial defences 38 Adherence to structures such as the ECM is therefore a key step in the development of disease The ECM consists of a complex mixture of macromolecules including collagens bronectin brinogen vitronectin laminin and heparin sulfate 38 all of which function as ligands for bacterial adhesion TRENDS in Microbiology Vol14 No3 March 2006 145 With the continued rise in antibiotic resistance in bacteria there is an urgent need to nd alternative ways to combat microbial and speci cally bacterial infection Inhibition of bacterial adhesion is one potential thera peutic approach but usually requires an intimate knowl edge of the adhesins involved in infection Phage display lends itself perfectly to the investigation of possible binding partners for a particular ligand and this application of phage display was rst exploited by Jacobsson and Mykberg 3940 to identify genes encoding bacterial proteins that interact with host proteins By constructing libraries from random fragments of bacterial genomic DNA ie shotgun phage display and subsequent panning against components of the host such as proteins of the ECM host serum or plasma it is possible to identify bacterial genes that encode cell surface adhesins Microbial proteins including cellbound and soluble proteins that bind to mammalian target proteins were termed receptins by Kronvall and J onsson 41 Following the initial description of geneIIIbased phage display to identify genes coding for ligandbinding domains of bacterial receptins by Jacobsson and Frykberg 39 many genes involved in host pathogen interactions from a variety of bacterial species have been discovered using phagedisplay systems Table 2 The majority of these studies have been carried out using geneVIIIbased vectors such as the pG8H6 or pG8SAET phagemid vectors developed by J acobsson and Mykberg 4042 Panning of pIIIbased libraries tends to select for fusion proteins with the strongest interaction with the ligand of choice but only a small fraction of p111 libraries contain functional inserts 43 Use of pVIII generates phage with multiple copies of recombinant proteins but the selection process tends to select for lower a nity interactions Shotgun phage display has also been used in the mapping of binding domains within proteins coded by genes of interest Instead of random fragmentation of genomic DNA from a particular species a previously identi ed gene of interest is fragmented by sonication and used to generate a singlegene phage display library This approach has been used to map bronectinbinding activity to two Cterminal domains of the bronectin binding protein FNZ of Streptococcus equi 44 the IgG Table 2 Identification of bacterial receptins by phage display screening Organism Vector Ligand quot 39 39 39 Refs Helicobacter pylori pG SSAET Plasminogen Pg b3 54 Staphylococcus aureus pHenl Human IgG Sbi 46 pGSSAET von Willebrand factor vWBp 55 pGSSAET Platelets FnBPA FnBPB 56 pGSSAET Fibronectin FnBPA 57 Staphylococcus epidermidis pGSSAET Fibronectin Embp 58 pGSSAET Fibronectin and osteoblast cell line Second fibronectin binding 59 MC3T3 E1 domain within FnBPA and FnBPB pG8H6 Fibrinogen be 60 Streptococcus agalactiae pG3H6 Fibronectin Sch 61 pGSSAET Fibrinogen Fgang FgagV2 FgagV3 62 Streptococcus dysgalactiae pG8H6 Fibro nectln DemA 63 Streptococcus equi pGSSAET Fibronectl FnBP 64 Staphylococcus lugdunensis pGSSAET von Willebrand factor VWBl 65 pGSSAET Fibrinogen Fbl 66 www5ciencedirectcom 146 TRENDS in Microbiology Vol14 No3 March 2006 and 32glycoproteinIbinding domains of the Sbi protein of S aureus 4245 and the IgG and albuminbinding domains in two cellsurface receptors MAG and AG from group C streptococci 46 The application of shotgun phage display to the identi cation of bacterial virulence factors has been further extended by Frykberg and colleagues with the development of phagemid vectors that only enable the display of bacterial exported proteins 4748 Many exported microbial proteins are adhesins enzymes or toxins all of which might have a role in pathogenicity The libraries constructed by the shotgun method might not achieve full coverage of bacterial genomes for two reasons i the low transformation ef ciency of E coli limits the number of primary clones ie those containing unique inserts and ii only one in 18 clones displays fusion proteins see earlier However despite these limitations the success of this method is obvious considering h numbers of shotgun phage display libraries that have been used to identify novel genes that encode putative bacterial receptins and protein binding domains Table 2 Us f this technique identi es multiple microbial genes encoding proteins that poten tially interact with the host in a single panning experiment and such results provide a starting point to determine the importance of various bacterial genes in pathogenesis It is likely that phage display NPLs will become even more widely used in the study of infectious diseases particularly because the growing number of available bacterial genomes means that the DN frag ments that encode phagedisplayed peptides and proteins can be easily mapped to a particular bacterial gene Concluding remarks The study of infectious diseases has bene ted greatly from phage display technology This technique has many advantages the link between phenotype and genotype the enormous diversity of variant proteins displayed within a single library and the exibility of selection that can be performed in vitro or in vivo Earlier limitations on this technique such as the unsuitability of pIII or pVIIIbased lamentous phage display systems for the display of cDNA coding for peptides or problems with protein conformation have been circumvented by continual development of the technology The versatility of phage display means that it can be adapted easily to many different areas of research as highlighted by the variety of biological questions to which phage display has been applied It has yielded interesting and important results in the study of infectious diseases not least in epitope mapping the identi cation of potential vaccine candidates and bacterial adhesins However the vast potential of phage display is such that there are many more ways of applying the technology to these areas of research There is certainly scope for developing new strategies to identify host receptors for microbial products by panning in vivo in animal models or against ex vivo tissues or organs There are numerous examples of synergistic relationships between opportunistic bacteria that could be examined by panning phage display libraries against each other to investigate interactions between microbes Perhaps www5ciencedirectcom the most exciting potential use of phage display in the study of infectious diseases is in comparative genomics The number 0 sequenced microbial genomes has increased greatly in the past ten years and continues to do so as more genomes are sequenced This will enable detailed bioinformatic analysis of genes identi ed by panning of NPLs and a comparison of the genes implicated in pathogenicity between groups of closely related bacteria Refere 1 mith GP 1985 Filamentous fusion phage novel expression quot r v the quot r pipmzz 1315 1317 2 Russel M et al 2004 Introduction 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