guidelines for membrane protein engineering derived from de novo designed model peptides

Li-Ping LiuCharles M. Deber Guidelines for MembraneDivision of Biochemistry

Research, Protein Engineering DerivedHospital for Sick Children,

Toronto M5G 1X8; from De Novo DesignedDepartment of Biochemistry, Model PeptidesUniversity of Toronto,

Toronto M5S 1A8,Ontario, Canada

Abstract: Notwithstanding great advances in the engineering and structural analysis ofglobular proteins, relatively limited success has been achieved with membrane proteins—due largely to their intrinsic high insolubility and the concomitant difficulty in obtainingcrystals. Progress with de novo synthesis of model membrane-interactive peptides presentsan opportunity to construct simpler peptides with definable structures, and permits one toapproach an understanding of the properties of the membrane proteins themselves. In thepresent article, we review how our laboratory and others have used peptide approaches toassess the detailed interactions of peptides with membranes, and primary folding at membranesurfaces and in membranes. Structural studies of model peptides identified the existence ofa ‘‘threshold hydrophobicity,’’ which controls spontaneous peptide insertion into membranes.Related studies of the relative helicity of peptides in organic media such as n-butanol indicatethat the helical propensity of individual residues—not simply their hydrophobicity—maydictate the conformations of peptides in membranes. The overall experimental results providefundamental guidelines for membrane protein engineering. q 1998 John Wiley & Sons,Inc. Biopoly 47: 41–62, 1998

Keywords: membrane protein engineering; de novo designed model peptides; helix propensity

INTRODUCTION resolution extremely challenging. Compared to over2000 globular proteins that have been characterized bymultidimensional nmr or x-ray crystallography, three-Whereas protein engineering is becoming an estab-dimensional structures at atomic level resolution havelished research tool for studying structure–functionbeen obtained for only a handful of membrane proteinsrelationships of soluble proteins and for improving(viz., bacteriorhodopsin,1 photosynthetic reactiontheir properties, the engineering of membrane proteinscenter, 2 light-harvesting complex II, 3,4 porin,5–8 he-is just coming of age. Membrane protein engineeringmolysin,9 and glycophorin A.10 It was found thatis less straightforward due to the paucity of structuralthe structures of the membrane-spanning segmentsdata on the membrane proteins themselves. Amongof these proteins tended to be relatively simple, withseveral factors, the high insolubility of membrane pro-

teins has made their structural characterization at high the majority of them adopting the a-helical confor-

Correspondence to: Charles M. Deber, Division of Biochem-istry Research, Hospital for Sick Children, Toronto M5G 1X8,Canada. E-mail: [email protected] (Peptide Science), Vol. 47, 41–62 (1998)q 1998 John Wiley & Sons, Inc. CCC 0006-3525/98/010041-22

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42 Liu and Deber

FIGURE 1 Representative structures of membrane proteins. (a) Bacteriorhodopsin (Brook-haven protein data bank, entry 2BRD, transmembrane a-helices) 1; (b) Porin (entry 3POR,transmembrane b-barrel) . 5

mation; a few are b-barrels (Figure 1). A clear BACKGROUNDmessage from these known structural data is thatintegral membrane proteins come in a very limited Protein Engineeringvariety of basic architectures, and that their de novo

With the development of genetic engineering technol-design should be comparatively simple—once weogy, the gene for essentially any protein found infind the rules. 11,12 Considering the variety of biolog-nature can be cloned. Combining these genetic modi-ical functions performed in vivo by membrane pro-fication techniques with known protein structural datateins in health and disease, it remains tempting tofrom x-ray crystallography and nmr, novel proteinsgenerate novel membrane proteins with desiredwith desired structures can be created, with improvedfunctions and structures, and to modify/ improve thefunctions and new properties that cannot be achievedproperties of existing systems.as effectively by any other methods.13,14 At present,Peptide synthesis allows a great deal of freedomproteins generated by engineering techniques havein the structural design of peptides, through system-shown remarkable promise in fields such as rationalatic variation in sequence and chemical modifica-design of drugs,15 enhancement of enzyme perfor-tion. The synthetic approach provides an alterna-mance in industrial processes,16 and recombinant im-tive path through which we can complement andmunotoxins for cancer therapy.17 However, these stud-extend our understanding of the dynamic struc-ies have been focused on the modification of existingtures and functions of protein segments in the lipidfunctions of globular proteins; examples in which newenvironment. The focus of this review is to presentfunctions have been engineered into membrane pro-how our laboratory and others use synthetic peptidesteins are rare due to the current limited understandingto model the membrane-embedded domains ofof their structures and functions.membrane proteins, and thereby to approach the

development of algorithms for the relationshipsamong primary sequence, secondary structure, and Engineering Membrane Proteinstertiary interactions in an environment so little ex-plored—the membrane. The information gained Membrane proteins generally are composed of

transmembrane domains (TM) and extramembra-from such peptide studies is expected to providefundamental guidelines for membrane protein engi- nous loops. The engineering of loop domains can

follow routes similar to soluble proteins; for in-neering.


Guidelines for Membrane Protein Engineering 43

stance, ligand-binding sites can be modified in order In previous studies, a series of peptides with a 10-residue hydrophobic core [typical sequence: H2N-to probe structure/function relationships, 18 and im-

munological /chemical tags can be added for the SKSKAXAAXAWAXAKSKSKS-OH, where XÅ an uncharged amino acid (excluding Trp andpurpose of purification and localization.19–21 Yet,

for TM domains, since the membrane environment Cys)] have been synthesized and studied in mem-brane mimetic environments. 28,47 The major resultsprovides a solvation matrix quite different from wa-

ter, many studies have suggested that the rules for may be summarized as follows: (a) the helicity ofmodel peptide in membrane environments corre-TM segment insertion, conformational preferences,

folding, and tertiary contacts are likely to differ lates with its segmental hydrophobicity, with therank order of helix formation for uncharged aminofrom those for soluble segments. For example, tradi-

tional helix-destabilizing amino acid residues (Gly acid in SDS micelles given as Ile ú Leu Ç Valú Met ú Phe ú Ala ú Gln ú Tyr ú Thr ú Serand Pro) can be well accommodated in TM do-

mains22–27; b-branched residues such as Val and Ile, ú Asn ú Gly; and (b) initial observations suggestthat a ‘‘threshold hydrophobicity’’ may exist forwhich strongly prefer the b-conformation in soluble

proteins, actually promote the a-helical conforma- stable partitioning of a peptide segment into thelipid matrix (see Ref. 47 for details) .tion when in a membrane environment.28

Inasmuch as most TM segments in proteins arecomprised of a continuous stretch of ca. 20 apolarUse of Peptides as Models for the Studyamino acids—longer than the 10-residue hydropho-of Membrane Proteinsbic segments contained in the peptides used in theabove studies—our lab went on to the design andNaturally occurring short polypeptides, fragments

of membrane proteins, and peptides designed de synthesis of a subsequent generation of model pep-tides containing a hydrophobic segment of 19 aminonovo have all been shown to mimic structural and

functional aspects of intact membrane proteins. For acids.48,49 The prototypical sequence of peptides isKKAAAXAAAAAXAAWAAXAAAKKKK-example, gramicidin, a 15-residue hydrophobic pep-

tide that forms well-defined ion channels in mem- amide,where X Å each of the 20 commonly oc-curring amino acids. The rationale for this peptidebranes, has served as a model for understanding

membrane protein folding, and ion channel structure design is given as follows:and function.29–31 Similar studies have also beenperformed with alamethicin,32–35 pardaxin,36–39 and 1. The hydrophobic segment of peptide is com-

prised of 19 amino acids, which when foldedsynthetic model peptides.28,40–42 Synthetic peptidescorresponding to the actual TM sequences of mem- into an a-helical conformation, is of sufficient

length to span a phospholipid bilayer (50).brane proteins have also been found to assembleinto their membrane-bound states; examples include 2. Distributions of the three guest residues ‘‘X’’

have been designed to preserve both angularthe transmembrane domain of glycophorin A,10 thechannel-inducing proteins phospholamban,43,44 and and longitudinal symmetry around the helix,

thereby minimizing any bias from amphi-minK.45,46 The above studies thus demonstrate thatpeptide studies have provided an alternative ap- pathic character that may arise when X were

substituted by polar and charged residuesproach, simple yet reliable, to systematically assessthe mechanisms involved in membrane protein fold- (Figure 2). This will guarantee that all pep-

tides approach the anisotropic membrane en-ing and the detailed protein interactions with lipidbilayers. vironment in an equivalent manner, without

preferred orientations. In addition, triple sub-stitutions of guest residue X in the hydropho-bic core amplify the effect of ‘‘guest’’ re-DE NOVO DESIGN AND SYNTHESIS

OF MEMBRANE-SPANNING PEPTIDES placements, ensuring that the spectroscopicmeasurements can detect their effect(s) .

3. Ala, the most appropriate background residueIn our laboratory, de novo designed model peptideshave been successfully utilized to improve the fun- as demonstrated by previous studies, 28,47 was

chosen as the ‘‘host’’ residue.damental understanding of protein–lipid interac-tions. The advantage of de novo design is that the 4. A Trp residue was incorporated into the hy-

drophobic segment as a fluorescence probe tostructure of models can be easily controlled; thus,there are fewer structural possibilities, simpler prop- monitor characteristics of the local microenvi-

ronment.erties, and more facile interpretation of the results.


44 Liu and Deber

FIGURE 2 Schematic representation of de novo designed peptides of prototypical sequenceKKAAAXAAAAAXAAWAAXAAAKKKKNH2, where X Å each of the 20 commonly oc-curring amino acids. (a) Proposed membrane-spanning a-helical conformation of the peptidewhere X Å Val; the micelle itself is not shown. Solid (green) spheres represent methyl groupsof Ala residues. Lys residues at peptide N- and C-termini are depicted as disordered (blue)‘‘tails.’’ The Trp indole side chain is shown in line form for clarity. (b) Helical wheel represen-tation (top view) of the peptide hydrophobic segment (residues 3–21), where the three Xresidues (gold) are distributed triangularly around the helical axis. Adapted from ref. 124.

5. Lys residues were added at N- and C-termini preferences that influence the stabilities of proteinsecondary structures and folding. Various predic-to enhance the water solubility of peptides.

Compared to Lys-Ser pairs used in previous tions regarding to the intrinsic conformational pro-pensities (a-helical and b-sheet) of the 20 com-design, a cluster of Lys residues should be

more effective in preventing peptide–peptide monly occurring amino acids in globular proteinshave been developed over the last two decades,aggregation through the resulting high local

positive charges. For the same purpose, the C- based largely on statistical and empirical analysis ofknown protein crystal structures, 51–53 experimentalterminus was amidated to eliminate potential

electrostatic attractions that might occur inter- measurements in host–guest model peptides,54–62

site-directed mutageneses of host proteins,63–65 andor intramolecularly.molecular dynamics calculations.66,67 Whereas inmembrane proteins many efforts have been madetoward identifying the hydrophobic stretches ofCONFORMATIONS OF PEPTIDES IN

AQUEOUS BUFFER VS IN NONPOLAR amino acid that likely traverse the membrane,68,69

little attention has been paid to addressing the con-SOLVENTSformational propensities of individual amino acidsin membrane environments, principally because theProtein secondary structures have been viewed as

fundamental building blocks for protein folding, a-helices have been found as the overwhelminglyfavored structural motif, at least in the few con-structure, and design. Previous studies indicate that

individual amino acids have distinct conformational firmed structures. 1,2,4,10 Do these results imply that



all amino acids have the same ‘‘helical propensity’’ by the Glu residue can be explained by the stabiliz-ing effect arising from salt-bridge formation be-once they have been deposited in the membrane?

The solubility of the present peptides in a wide tween the negatively charged Glu residues nearestthe peptide termini with the positively charged Lysvariety of media has made it feasible to investigate

and compare specifically the conformational prefer- residues at the neutral pH. For the same reason, Aspalso shows a fairly high, perhaps overdetermined,ences of the various residues in aqueous buffer vs

membrane-mimetic media [lipid micelles, smaller a-helical propensity.Consistent with many other studies, Pro and Glyunilamellar vesicles (SUVs) and organic solvents] .

In these studies, the aggregation states of peptides possess the lowest helix propensity determined inthis series of peptides, which is mainly caused bywere monitored by CD measurements and by size

exclusion high performance liquid chromatography the rigid backbone of Pro and the flexible backboneof Gly. Although b-branched amino acids such as(HPLC), from which it was found that through the

concentration range of 5–250 mM, peptides re- Val, Ile, and Thr are normally taken as a-helixbreakers since their x1 side-chain dihedral angle ismained monomeric in all experimental media.severely restricted in the a-helix,73,74 a fairly highhelical propensity of Ile was observed in this work.

a-Helical Propensities of IndividualA similar result was also obtained in a T4-lysozymeAmino Acids in Aqueous Solutionstudy.64 It has been known that the loss of side-chainentropy and the formation of side-chain hydrogenThe helix propensity of a particular amino acid is

a measure of how its side chain influences the con- bonds destabilize helix formation, while hydropho-bic interaction between apolar side chain atoms andformation of the peptide backbone. The propensity

arises from relatively short-range interactions of the the peptide backbone stabilize the helix formation.For Ile, although the loss of side-chain entropy op-side chain with the peptide backbone and solvent,

and from interactions of the peptide backbone with poses helix formation, burial of the nonpolar surfaceof the Ile branched chain favors helix formation;itself and solvent. Since the only difference among

the present peptides is the guest X residues, it is this may explain, in part, why the helix propensityof Ile is significantly greater than that of Val.64reasonable to assume that variations in secondary

structures observed among these peptides are attrib- Ala possesses the second highest helix propensityin this work (and the highest in some other sys-utable to the properties of the X residue. Hence, the

difference in peptide helicity can be interpreted as tems51,63,66,71 due to the following reasons: (1) thereis no loss of side chain entropy upon helix forma-a manifestation of the propensity for each X-amino

acid to form an a-helical structure in a specific envi- tion, and (2) its small nonpolar side chain can par-ticipate in hydrophobic interactions with the peptideronment.

Peptide conformation was first measured in aque- backbone to stabilize the helix conformation.ous buffer. CD, which is sensitive to secondarystructure of proteins and polypeptides in solution,

a-Helical Propensities of Individualwas used to monitor the conformations of model Amino Acids in Nonpolar Solventspeptides. In aqueous buffer, peptides formed partialor nonhelical conformations as shown by u222 data in Fatty acyl chains of lipid molecules present a hy-

drophobic environment to proteins that are embed-Table I, and CD spectra shown for selected peptides(Figure 3). In water, a generally good correlation ded in membranes. This environment provides a

very different solvation matrix than water, and con-was obtained when comparing the present peptidehelicities with Pa values predicted by Chou and sequently, proteins in membranes tend to fold in

different manners than proteins in an aqueous mi-Fasman (Figure 4). When the helical propensitiesof individual amino acids determined with this pep- lieu. Organic solvents, with dielectric constants be-

tween pure water and the hydrocarbon interior oftide model were also compared with those obtainedin other experimental systems,51,63,70–72 there is a biological membranes, have been used in several

studies to mimic the nonpolar environments ofgood agreement in both the rank order (Table II)and magnitude of the helical propensities (Table membranes.25,45,75–78 One of the main advantages of

organic solvents is that they create a quasi-membra-III) with the correlation coefficients ranging from0.67 to 0.88. These results imply that the present nous yet homogenous (isotropic) environment. Ac-

cordingly, certain of the complexities involved inpeptide model is an appropriate model to derive thehelix propensities of each amino acid. peptide–lipid interaction such as phase partitioning,

and electrostatic attractions between oppositeThat the highest helical propensity was displayed


46 Liu and Deber

Table I Helicity of Model Peptides H-KKAAAXAAAAAXAAWAAXAAAKKKK-Amide in Various Mediaa

0u222 1 1004 (degrcm2/dmole)

X Residue Aq.b n-Butanol SDSc LPGc LPCc

A 2.06 3.72 3.58 3.65 3.59C 0.91 3.28 3.50 3.60 3.51D 1.47 2.66 2.63 2.98 1.61E 2.25 2.54 2.88 2.89 2.04F 1.12 3.78 3.53 3.60 3.58G 0.34 3.44 3.01 3.02 0.62H 0.79 2.90 2.32 2.60 1.16I 1.54 3.88 3.79 3.54 3.51K 1.12 2.65 2.20 2.5 1.24L 1.80 3.84 3.72 3.78 3.75M 1.33 3.67 3.53 3.55 3.39N 0.59 2.82 2.42 2.58 0.98P 0 1.70 1.14 1.45 0.03Q 1.19 2.87 2.60 2.70 1.26R 1.35 2.84 2.65 2.92 1.35S 0.79 2.99 2.88 2.90 1.20T 0.90 3.27 3.36 3.22 1.62V 1.03 3.82 3.67 3.70 3.68W 1.13 3.20 3.16 3.09 3.03Y 1.16 3.33 3.22 3.30 3.20

a The u222nm in CD spectra is used as a measure of helicity. Peptide concentration Å 30 mM.b Aq., aqueous buffer Å 10 mM Tris HCl, 10 mM NaCl, pH 7.0.c Micelle concentration Å 10 mM SDS, LPG, or LPC in aqueous buffer.

charges of peptides and lipid head groups, can be can solubilize all 20 peptides, neither of the abovetwo solvents could be used. A practical choice wasignored. Also, while membrane proteins/peptides

are typically hydrophobic, and virtually insoluble n-butanol, a moderate nonpolar solvent with a di-electric constant of 17.8 at 257C.in water, they tend to be quite soluble in many

organic solvents. Furthermore, working with or- In n-butanol, peptides formed predominantly a-helical conformations, as demonstrated by CD spec-ganic solutions as opposed to lipid vesicles greatly

simplifies the application of various spectroscopic tra that possess the characteristic double minima at208 and 222 nm (selected peptides shown in Figuretechniques owing to the stability and smaller size

of the ‘‘solvent’’ molecules, and generally the clar- 5) . In order to avoid any bias that might be causedin CD measurements by the limited solubility ofity of the solutions themselves. For these reasons,

a variety of organic solvents ( trifluoroethanol, some peptides in n-butanol, the effect of concentra-tion on the CD spectra of selected peptides wasmethanol) have been used in numerous studies to

obtain structural information of peptides/proteins in examined, as presented in Figure 6. CD spectrademonstrate that conformations for both nonpolarnonpolar environments. 36,76,79–84 It should be noted,

however, that some organic solvents could cause (Ala and Met) and polar (Asn and Arg) peptides areconcentration independent, and thus that the helicitydenaturation by disrupting the tertiary structures of

membrane proteins.85 differences observed among the peptides are notcaused by their differential solubility, but by theirThe question as to which organic solvent(s) most

closely resemble the environment of the interior of intrinsic propensities to form the a-helical confor-mation in a nonpolar environment. In order to assessa phospholipid bilayer has been raised. Several in-

vestigators have argued that octanol is the best the general applicability of the above experimentaldata, we used the mean residue ellipticity (u222)model solvent for a membrane, whereas others insist

that a purely nonpolar solvent such as hexadecane values for further quantitative analysis. Several re-searchers have analyzed the membrane inclusionis more appropriate (for a review, see Ref. 86). In

the present work, since we require a solvent that preference of individual amino acids based on



and qi is the relative frequency of occurrence of theamino acid i in a particular structural class.

Since in our model systems the guest residues Xare located in the middle of helix, we comparedthe experimental measurement helicity with the dataderived from the ‘‘helix middle.’’ For comparisonwith the conventional Chou–Fasman prediction, weconverted Jones’ log likelihood data si toPa(TM) —preference of an individual amino acidfor TM helices, where Pa(TM) Å qi /pi . The valuesof Pa(TM) derived from the helix middle of single-and multispanning membrane proteins are listed inTable IV. There is a good correlation (r Å 0.9 whenPro was excluded) between the u222 (n-butanol) andthe Pa(single-TM) (Figure 7). Similarly, Samateyet al. analyzed the periodic distribution of residuesin the sequence of 469 putative TM a-helices fromeukaryotic plasma membrane polytopic proteinswith correlation matrices.90 The distribution of resi-dues in the lipid-exposed surfaces of TM helicesalso show that Ile, Leu, and Val residues have highcompositions.

The above experimental data also appear to besupported by the observations obtained from mem-brane proteins. For example, although Gly and Proare among the most destabilizing residues for a-

FIGURE 3 CD spectra of selected model peptides inhelices in globular protein and random-sequenceaqueous buffer. The peptides shown (A, D, F, G, I, L,polypeptides,52,54,63,70 they do display a considerableS, and T) represent a diversity of residue hydrophobicitytendency to form a-helices in membrane environ-and side-chain chemistry. Peptide concentrations werements (see Table I) . Li et al. also found that in atypically 30 mM. The aqueous buffer was prepared frommembrane environment, a suitably placed Pro tends10 mM Tris HCl, 10 mM NaCl, pH 7.0. Curves reportedto protect a-helical structures rather than breakare based on triplicate measurements; standard deviation

is {1%. Adapted from ref. 124. them.25 Based on these data, it should not be unex-

known membrane proteins87–89—similar to whatChou and Fasman carried out for globular proteins.For example, Jones et al. 89 used a dynamic program-ming algorithm to provide a membrane topologymodel from single sequence information. This tech-nique is based upon statistics from well-character-ized membrane proteins to elicit the likelihood thata given amino acid residue will occur in any of fivedifferent types of locations: extramembranous loops(inside and outside the cell) , inside helix end, helixmiddle, and outside helix end. For each of the 5structural classes, log likelihood ratios (si) for eachof the 20 amino acids were calculated:

si Å ln(qi /pi)

FIGURE 4 Correlation of peptide helicity in aqueouswhere pi is the relative frequency of occurrence of solution with the Chou–Fasman prediction parameter

(Pa) . 51 Adapted from ref. 125.the amino acid i in all the sequences in the data set,


48 Liu and Deber

Table II Rank Order of Helix Propensities of Individual Amino Acids Obtained from Various Systems

Experimental,Experimental, Peptides Protein Statistical

Monomeric T4 LysozymeConsensusa Present Work Helicsb (Site 44)c Helixd Pae AGADIRf

A E A A E E AR A R L A M RL L L I M A KK I K M I L LM D E K R K MQ R M Q K F QE M Q R L Q EI Q I Y Q I IF Y Y V F W FW W C E V V WS F H F W D YY K S H Y H VH V W S D R SD C D T H T TC T F W C S DN H N C N C CV S V D S Y NT N T N T N HG G G G G G GP P P P P P P

a For details, see Ref. 123.b For details, see Ref. 70.c For details, see Ref. 64.d For details, see Ref. 71.e For details, see Ref. 52.f For details, see Ref. 72.

pected that one finds Gly and Pro quite commonly evidence of the existence of such a conformation,viz., Thr87 hydroxyl amide protons form a bifur-in native transmembrane helices, especially Pro in

transporters. 22,23,91 cated hydrogen bond with Gly83 carbonyl oxygenof its own monomer.10Similarly, Ser and Thr also occur frequently in

TM helices, as has been noted in many proteins.1,2,10 Sternberg and Gullick95,96 have proposed thatGlu, Gln and Asp could form hydrogen bondsHypotheses put forth suggest such that these resi-

dues might satisfy their hydrogen-binding capacity within the membrane in their protonated forms. Inhydrophobic membrane environments, the pKa forby forming H bonds with the carbonyl oxygen in

the preceding turn of the a-helix.92,93 A subsequent the acidic amino acid is raised such that at neutralpH, a proportion of the residues will be unionized.97survey performed by Gary and Matthews94 showed

that approximately 70% of Ser residues and at least An alternative arrangement has been observed inthe crystallographically determined structures of a85% of the Thr residues in helices form intrahelical

H bonds with the carbonyl oxygen atoms in the few membrane proteins, in which the TM segmentsare b-strand arranged as b-barrels5,9,98–101; some ofpreceding turn of the helix. H bonding within a

helix thus provides a route through which Ser, Thr, these proteins are listed in Table V. Thus, to get acomplete picture of secondary structural preferenceand Cys can satisfy their H-bonding potential, per-

mitting such residues to occur in helices buried (a or b conformation) of each amino acid in mem-branes, we performed an analysis parallel to thatwithin a hydrophobic milieu. Recent nmr results on

peptides corresponding to the TM segment of the used by Jones et al., where the relative occurringfrequency of each amino acid in the b conformationsingle-spanning protein glycophorin A provide clear



Table III Comparison of Helix Propensities of Individual Amino Acids in Aqueous Media Determined byVarious Methods

Experimental,Experimental, Peptides Protein Statistical

0u222 1 1004 (Aq.) Monomeric T4 LysozymeAmino Acid Present Worka Helicesb (Site 44)c Helixd Pae AGADIRf

A 2.06 0 0 0 1.42 0C 0.91 0.8 0.5 0.5 0.7 0.6D 1.47 0.9 0.5 0.4 1.01 0.59E 2.25 0.5 0.4 00.3 1.51 0.34F 1.12 0.9 0.4 0.3 1.13 0.47G 0.34 1.9 1 0.7 0.57 1.1H 0.79 0.8 0.4 0.4 1 0.62I 1.54 0.7 0.1 0.1 1.08 0.35K 1.12 0.4 0.2 0.2 1.16 0.15L 1.80 0.3 0 0.2 1.21 0.19M 1.33 0.5 0.1 0 1.45 0.21N 0.59 0.9 0.6 0.5 0.67 0.6P 0 0.57Q 1.19 0.6 0.2 0.2 1.11 0.32R 1.35 0.2 0.2 0.1 0.98 0.06S 0.79 0.8 0.4 0.5 0.77 0.52T 0.90 1.3 0.4 0.5 0.83 0.57V 1.03 1 0.3 0.3 1.06 0.51W 1.13 0.8 0.4 0.3 1.08 0.47Y 1.16 0.7 0.2 0.3 0.69 0.47

Correlationcoefficient 1 0.72 0.67 0.88 0.84 0.72

Values based on amolar ellipticity (present work); b,c,d,ffree energy (DG) relative to Ala; and eoccurrence frequency Pa.b For details, see Ref. 70.c For details, see Ref. 64.d For details, see Ref. 71.e For details, see Ref. 52.f For details, see Ref. 72.

was obtained (Table IV). Figure 8 shows the com- sults may shed some light on the factors that under-lie prediction of a-helices in membrane proteins.parison of Pa derived from TM helices of both sin-

gle- and multiple-spanning membrane proteins with This may allow us to get a clearer picture of wherehelices are highly probable rather than simply thePb from b-strands of b-barrel membrane proteins.

There is essentially no relationship. location of highly hydrophobic segments in the pro-tein primary sequence.The generally good correlation between the ex-

perimentally determined helical propensity for indi-vidual amino acids and their nonrandom occurringfrequency in transmembrane helices suggests that PEPTIDES IN LIPID MICELLESthe high frequency of occurrence in membranes ofresidues such as Leu, Val, Ile, and Phe derives not Lipid micellar systems have been widely adopted as

mimics of biological membranes.10,102–104 Typically,only from their hydrophobicity, but also their intrin-sic propensity to form the a-helical conformation micelles consist of a hydrocarbon core, enclosed

by polar groups at the particle surface serving toin nonpolar environments. Since the prediction ofmembrane protein folding is one of the most urgent maintain solubility in water. Micelles provide a sim-

ilar anisotropic environment to lipid vesicles, but intasks in life sciences—and most previous studieshave focused on the prediction of hydrophobic the laboratory they are easier to prepare and manip-

ulate. Therefore, lipid micelles have been employedstretch by hydropathy plots alone—the above re-


50 Liu and Deber

FIGURE 5 CD spectra of selected model peptides in pure n-butanol. Peptide concentrationsÅ 30 mM. Curves reported are based on triplicate measurements; standard deviation is {1%.

widely to study peptide/protein–lipid interactions. incorporation of proteins/peptides into micellesshould be applicable to the penetration of proteinsExamples include anionic micelles such as sodium

dodecylsulfate (SDS),28,105 deoxycholic acid into membranes, and their resulting conformationshighly representative of the natural situation.(DOC),106 and lysophosphatidylglycerol (LPG)48,49;

cationic micelles such as cetyldimethylethylam- Table I lists the mean residue molar ellipticity at222 nm of each peptide in SDS, LPG, and LPCmonium bromide (CTAB) 107,108; nonionic micelles

such as b-octylglucoside (b-OG) 109; and zwitter- micelles. As can be seen from these data, all pep-tides (excluding Pro) display predominantly a-heli-ionic micelles such as lysophosphatidylcholine

(LPC).49,80 Among these systems, SDS, LPG, and cal conformations in SDS and LPG micelles,whereas in LPC micelles, only some peptidesLPC are used most frequently owing largely to their

relatively small size (i.e., for nmr studies where formed helices, while others showed essentially ran-dom structures virtually unchanged from those ob-dynamics are key); while both LPG and LPC mi-

celles possess phospholipid head groups identical served in aqueous media. Comparison of CD spectrain n-butanol, SDS, and LPG of Asn and Tyr peptidesto those found in natural membranes. Overall, the

interfacial environments created in micelle head (Figure 9) showed that, in varying media, the meanresidue molar ellipticities at 222 nm of a given pep-group regions simulate very closely those in biologi-

cal membranes. Thus, information derived from the tide differ slightly, i.e., the value of 0u222 in n-



FIGURE 6 CD spectra of peptides with X Å Ala and X Å Arg, at varying concentrationsin pure n-butanol. Peptide concentrations range from 7.5 to 120 mM. Curves reported are basedon triplicate measurements; standard deviation is {1%. Similar CD spectra were also obtainedwith Met and Asn peptides.

butanol ú LPG ú SDS. These variations may be with LPG micelles also enhances the helical contentof hydrophilic peptides.caused by the following reasons: (a) the polarity of

The presence of the Trp residue in the middle ofpure n-butanol may be lower than the hydrocarbonthe hydrophobic segment of each peptide providesinterior of SDS and LPG micelles, as many studiesanother practical method to monitor the peptide–have demonstrated that micellar and membranousmicelle interaction. In an aqueous environment, theinteriors are not purely hydrophobic, but that thereemission maximum of Trp is ca. 350 nm, but whenis ca. 5% content of water110,111; and (b) the lowestit transfers to a relatively nonpolar environment, ahelix percentage in SDS micelles may be due to theblue shift in emission maxima concomitant with(partial) denaturing effect of the monomeric SDSintensity enhancement occurs. Table VI lists themolecule.blue shift values of all peptides in various mediaUnlike that in LPG and SDS micelles, in LPC(SDS, LPG, and LPC). In agreement with the abovemicelles the peptides are segregated into two dis-observation of relative peptide helicity, there is antinct groups, shown for selected peptides in Figureca. 10–15 nm blue shift for peptides in SDS and10. To determine the origin this distinction, FigureLPG micelles, but for peptides in LPC micelles, the11 was plotted to accommodate peptide helicity inblue shift is observed only for those that adoptedaqueous buffer, LPG and LPC micelles. The diago-an a-helical conformation.nal represents an ideal correlation, which would be

expected if peptide helicity is identical in the eachcorresponding environment. For LPC vs water, rela-

DETERMINATION OF THE THRESHOLDtively hydrophilic peptides (where X Å D, E, G, H,HYDROPHOBICITY WITH MODELK, N, P, Q, R, S, T, respectively) behave similarly,PEPTIDESbut LPC can increase the helical content of rela-

tively hydrophobic peptides (X Å A, C, F, I, L,M, V, W, and Y, respectively) . For LPC vs LPG, The notion that there is a hydrophobicity threshold

for stable integration of transmembrane segmentshydrophobic peptides behave similarly, but binding


52 Liu and Deber

Table IV Structural Preferences (a and b) of nines, Kuroiwa et al. found that efficient membraneAmino Acids in Membranes anchoring required a stretch of 19 or more Ala’s,

but only 9 or more Leu’s.115 With a 50 : 50 mixturePa (mid)a

of Leu and Ala, 11 residues or more were required.Similarly, Chen and Kendall found that efficient

Amino Acid Singlestop-transfer function and membrane anchoring inResidue Spanning Multispanning Pb

b

the E. coli inner membrane of a 21-residue segmentrequired a minimal hydrophobicity, as representedAla 1.86 1.25 1.16by a mixture of 7 Leu : 14 Ala.116 However, whileArg 0.08 0.09 0.85the threshold hydrophobicity was quantified in eachAsn 0.10 0.61 0.60

Asp 0.06 0.26 0.62 specific biological system in the examples above,Cys 0.76 1.43 0.00 elucidation of the general principles involved re-Gln 0.11 0.28 0.87 mains to be determined. Our observation that modelGlu 0.05 0.15 0.86 membrane-interactive peptides become segregatedGly 1.36 1.00 1.03 into two distinct groups upon interaction with LPCHis 0.27 0.36 1.07 micelles ( that is, no partially inserted peptide exists)Ile 2.69 1.63 1.28

stimulated us to quantify the threshold hydropho-Leu 2.53 1.48 1.21bicity requirement using the present peptide model.Lys 0.08 0.12 0.79To approach the quantitation of this minimal hydro-Met 1.26 1.22 1.23phobicity requirement, we first constructed a hydro-Phe 1.63 1.72 1.13phobicity scale from relative retention times ob-Pro 0.30 0.79 0.17

Ser 0.63 0.88 0.82 tained from reversed-phase HPLC experiments.Thr 0.70 0.93 1.03Trp 0.45 1.30 0.96

HPLC-Derived Hydrophobicity ScaleTyr 0.39 0.90 1.53Based on Hydrophobic PeptidesVal 2.32 1.55 1.37

Hydrophobicity scales are among the most abun-a Pa was derived from the data of si reported in Ref. 89; seedant scales to have been developed in protein sci-text for details.

b Pb was derived from structural analysis of residue occur- ence; no less than 82 scales were collected in arences in b-barrel-forming proteins (Table V). review by Nakai et al. in 1988. 117 This prolifera-

tion of hydrophobicity scales arose partially from

has long been proposed, viz. an all-or-nothing char-acteristic in biological systems that dictates the loca-tion of protein/polypeptide segments. For exam-ples, Qa-2—a cell surface glycoprotein anchored byphophatidylinositol—was converted to an integralmembrane protein by a single amino acid mutationof hydrophobic segment (Asp to Val) . 112 Similarly,the mutation of Ile8 to Asn of OmpA signal se-quence removed its ability to insert into modelmembranes.113 Using genetic and molecular tech-niques, Lee and Manoil performed a study of thesequence requirements for the first membrane-span-ning sequence (TM1) of Escherichia coli serinechemoreceptor to promote protein translocation.114

They found that maintaining a minimum hydropho-bicity value is essential for efficient protein translo-cation.

Many efforts have been made to establish theuniversal requirements for threshold hydropho-bicity. Using dog pancreas microsomes and in vitro FIGURE 7 Correlation of peptide helicity in n-butanoltranslation of a secretory protein into artificial stop- with Pa (TM) derived from midsegments of single span-

ning membrane proteins. 89 Adapted from ref. 125.transfer sequences composed of leucines and ala-



Table V b-Barrel-Forming Membrane Proteins Used to Obtain Pb

Swiss-Prot StructuralCode Proteins Characterization Reference

P31243 PORI_RHOCA X-ray (1.8 A) 98P02931 OMPF_ECOLI X-ray (2.4 A) 101P02943 LAMB_ECOLI Topology 100P09616 HLA_STAAU X-ray (1.9 A) 9P39767 PORI_RHOBL X-ray (1.96 A) 99

the diverse definitions of hydrophobicity. There with the result being a relatively longer retentiontime. In contrast, the hydrophilic peptides’ reten-are at least three distinct meanings 118: (a ) hydro-

phobicity has been used to refer to the transfer of tion times are relatively shorter. Therefore, anHPLC-based scale serves to define the overall hy-a nonpolar solute to any aqueous solution, (b)

hydrophobicity has been used more specifically to drophobicity of a peptide directly as its relativeefficacy of interaction with another (standardized)refer to transfer of polar solutes into a nonpolar

solution, and (c ) hydrophobicity has been used hydrophobic surface. This latter situation typifiesthe circumstances a hydrophobic TM segmentto refer to particular molecular models generally

involving the ordering of water molecules around would encounter in the midsection of a biologicalmembrane.the nonpolar solutes. Since the retention time of

a given peptide interacting with an HPLC support About 20 years ago, Porath et al. first proposedthat the hydrophobic surface in reverse phase HPLCis based on the competing attraction between the

peptide molecule and the solvent for the nonpolar may be a useful probe for investigating the behaviorof peptides and proteins at a hydrophobic interface,stationary phase as peptides pass through the col-

umn, the hydrophobic molecules interact more such as a lipid bilayer. 119 In 1986, Parker et al.presented the first full set of hydrophilicity parame-strongly with the hydrophobic stationary phase,

FIGURE 8 Comparison of conformational preference (a- or b-) of individual amino acidsin membrane environments of (a) Pb (TM) vs Pa (single-TM), and (b) Pb (TM) vs Pa (multi-TM).


54 Liu and Deber

FIGURE 9 CD spectra of X Å Asn and X Å Tyr peptides in pure n-butanol, SDS, and LPG.Peptide concentrations Å 30 mM. Micelle concentrations: 10 mM SDS or LPG in 10 mM TrisHCl, 10 mM NaCl, pH 7.0. Curves reported are based on triplicate measurements; standarddeviation is {1%.

ters derived from HPLC,120 which was based on the water, and B is 0.1% TFA/acetonitrile. If we as-sume that the retention time of a given peptide isretention times of 20 model synthetic peptides, Ac-

Gly-X-X-(Leu)3-(Lys)2-amide (X Å each of the directly related to the summed hydrophobicity/hy-drophilicity of the amino acids in that peptide, thecommonly occurring amino acids) . They found that

surface sites of a protein predicted by the HPLC experimentally measured retention times of eachpeptides can be converted to a relative hydropathyhydrophilicity scale correlated remarkably well with

the known antigenic sites for this protein and acces- index for the corresponding substituted X residues.The following equation was used in the data conver-sible sites as determined by x-ray data. In further

studies of peptides designed to model coiled-coil sion:conformations, the same laboratory investigated twoseries of de novo designed amphipathic a-helical

H Å 10 1 DtRX-Lys /DtRPhe-Lys 0 5.00peptides, AX9 (Ac-EAEKAAKEXEKAAKEAEK-amide), and LX9 (Ac-ELEKLLKEXEKLLKE-LEK-amide), to examine how the hydrophobic en- where DtRX-Lys is the retention time difference be-

tween the X peptide and the most hydrophilicvironment affected the relative hydrophilicity/hy-drophobicity of amino acid side chains.121 ‘‘Lys’’ peptide, and DtRPhe-Lys is the retention time

difference between the most hydrophobic ‘‘Phe’’In the present work, we constructed a novel hy-drophobicity scale based for the first time on the peptide and ‘‘Lys’’ peptide. Through these calcula-

tions, the most hydrophobic amino acid residue,retention times of the series of hydrophobic modelpeptides. Peptide retention times were recorded on Phe, was assigned a value of /5, while the most

hydrophilic one, Lys, with a value of 05. The re-a reversed-phase Primesphere C4 column using alinear AB gradient (2% B/min) elution at a flow sulting values are listed in Table VII.

Comparing the retention times of the present pep-rate of 1 mL/min, where eluent A is 0.1% TFA/dd



1. The hydrophobicity assignment of the aro-matic residue Trp has the greatest variability,i.e., ranging from very hydrophobic (HPLC-based scale) , moderately hydrophobic (GESscale) to hydrophilic (K-D scale) . In gen-eral, one finds the largest differences amongvarious polarity scales for those amino acidsthat have polar character but uncharged sidechains. Even though Trp has the largest sur-face area and therefore the largest hydropho-bic component of all the amino acids, thehydrophilic component can move it to alower position on any polarity scale that as-sumes total immersion of the side chain ina nonpolar phase. However, in the presentsituation (viz., the HPLC-derived scale) , theTrp residue is presented to an interface whereits hydrophilic interactions are not disrupted,and therefore its hydrophobic character be-comes apparent.

2. That both Glu and Asp were assigned a rela-tively higher hydrophobicity value in theHPLC-derived scale was probably due to thedeionization of acid side chains ({COOH)under the prevailing experimental conditions

FIGURE 10 CD spectra of selected model peptides in (0.1% TFA).LPC micelles. Peptide concentrations Å 30 mM. LPCmicelle concentration: 10 mM in 10 mM Tris HCl, 10

Threshold HydrophobicitymM NaCl, pH 7.0. Curves reported are based on triplicatemeasurements; standard deviation is {1%. Adapted from Since under neutral conditions the net charge ofref. 124.

zwitterionic LPC micelles is zero, these micellescan be used directly to establish the requirementfor hydrophobicity. Through correlation of the CD-determined segregation of peptides in LPC micellestide series with the AX9 peptides reported by Sereda

et al., 121 a remarkably good correlation (r Å 0.97; (Figure 14) with the HPLC-derived hydrophobicityscale, we were able to define a threshold hydropho-Figure 12) was obtained. Yet, significant factual

differences exist between the two sets of peptides bicity, at ca. 0.4 (calculated as the mean residuehydrophobicity of the X Å Ala peptide, which con-with respect to their relative length (25 residues vs

18 residues); intrinsic character (nonamphipathic tains 18 Ala’s and one Trp in its hydrophobic core) ,above which peptide segments spontaneously inte-vs amphipathic); and number of guest residues (3

vs 1). As well, the differing chromatographic condi- grate into LPC micelles. If calculated by the fre-quently used hydrophobicity scale of Kyte and Doo-tions used to investigate their properties. The good

correlation may arise, in part, from the similar hy- little, 69 the numerical value of this threshold hydro-phobicity is 1.7.drophobic interaction between the peptide hy-

drophobic face (i.e., one X residue in a cluster of The biological relevance to proteins of this pep-tide-based experimentally determined threshold hy-Ala residues) and the stationary phase of the column

available to both series of peptides. In any case, the drophobicity was evaluated by examining mean res-idue hydrophobicity (calculated by the hydropathyresults provide evidence that HPLC retention times

represent a very reliable method for measuring the scale in the present work; see Table VII) for severalthousand helices using data bases derived from TMintrinsic hydrophobic character of the substituted X

residues. segments of single- and multispanning membraneproteins122 and from non-TM helices (non-Comparing this HPLC-based scale with two

other frequently used scales, i.e., the K-D 69 and transmembrane a-helices from globular and mem-brane proteins; Figure 15a). From the resulting dis-GES scale 68 (Figure 13) , we found the following :


56 Liu and Deber

FIGURE 11 Peptide helicity (measured by CD ellipticity values at 222 nm) in LPC micellesvs corresponding values in aqueous buffer (right ordinate), filled squares; and LPG micelles( left ordinate), filled circles . Values reported are based on triplicate measurements; standarddeviation is {1%. The diagonal represents an ideal correlation, which would be expected ifpeptide helicity is identical in the corresponding environment.

tributions of mean residue hydrophobicities, we dropathy scale69—which is derived largely fromconsiderations of ‘‘buried’’ vs ‘‘exposed’’ residuesfound that ú97% of single-spanning TM segments,

and ú96% of those comprising multispanning in a sample of crystalline globular proteins—gavebroadly comparable results for single-spanning TM(polytopic) membrane proteins, are above the cal-

culated threshold. Thus, the parameters for mem- segments, of which 84% were above the threshold(Figure 15b). However, while only 52% multispan-brane insertion established for model peptides can

be applied to natural TM segments contained in ning TM segments were above the threshold, essen-tially no non-TM helices (less than 2%) were aboveboth single- and multispanning membrane proteins.

When a comparative analysis was performed on 339 it. Thus, by using the present HPLC-derived scale(which identifies TM segments) in conjunction withnon-TM helices of length ¢ 19 residues, 22% were

above the threshold. As mentioned in the Materials the K-D scale (which identifies non-TM helices) ,one is able to distinguish a TM segment—or deand Methods, most of the non-TM helices are short

(average Å ca. 10 residues) . When this length fac- novo design a TM segment—readily and accuratelybased on the experimentally-determined thresholdtor is taken into account, less than 2% of the total

collected non-TM helices fulfill the requirements of hydrophobicity.both length¢ 19 residues and hydrophobicity abovethe threshold. Hence, the length of a helix also ap-pears to play a major role in determining the ability CONCLUSIONSof the segment to partition into the membrane envi-ronment. By using a set of de novo designed Ala-based pep-

tides, we were able to compare the a-helical propen-Parallel analysis carried out with the K-D hy-



Table VI Blue Shifts of Tryptophan Fluorescence competent for spontaneous membrane insertion asEmission Maxima (Dlmax) for Model Peptides in long as their average segmental hydrophobicity ex-Different Mediaa

ceeds the threshold; this latter phenomenon is ob-served, for example, in several of the seven TM

Dlmax Dlmax Dlmax segments of the multispanning transport proteinPeptide (SDS) (LPG) (LPC)

bacteriorhodopsin, where highly nonpolar residuescompensate for charged residues.1 That the mini-A 9 10 8mum hydrophobicity requirement is satisfied byC 15 10 8ú96% of over 5000 transmembrane segments de-D 8 7 0rived from a database of single- and multispanningE 11 11 0

F 16 12 10 intrinsic membrane proteins provides direct evi-G 13 12 0 dence that threshold hydrophobicity is the groundH 11 10 1 rule followed by most TM helices. These findings,I 14 12 10 in turn, relate the concept of threshold hydropho-K 8 10 1 bicity to the selective incorporation of transmem-L 14 12 10 brane segments into the lipid bilayer during the bio-M 12 12 10

synthetic translocation process without the require-N 8 7 0ment of any additional expenditure of energy.P 10 12 0

Given the central roles of membrane proteins inQ 8 10 0cellular processes ranging from nutrient uptake toR 10 10 0cell–cell communication, efforts to understand andS 10 10 0

T 8 10 0 control their structures are vital. The rational designV 12 11 9 of artificial proteins with purposely modified prop-W 8 9 8Y 9 11 11

Table VII Relative Hydrophobicity of Amino Acida lmax Å wavelength at which Trp fluorescence emission is Residues Determined by Retention Times in

maximal. For all peptides, lmax(aq.) Å 350 { 1 nm. Blue shift Reverse-Phase HPLCfor Trp emission: Dlmax Å Trp lmax(aq.) 0 Trp lmax (micelle).Excitation: 280 nm.

RelativeHydrophobicity

Model Peptide, Retention Time, of Guest Aminosities for individual amino acids in polar vs in nonpo- X Residue mina Acid Xlar environments. The generally good correlations ofexperimental data [i.e., peptide helicities in different F 22.58 5.00

W 22.49 4.88solvents (aqueous buffer and n-butanol)] with statis-L 22.40 4.76tical parameters (Pa derived from globular proteinsI 22.13 4.41and membrane proteins, respectively) suggest thatM 21.23 3.23the Pa is primarily a measure of the intrinsic confor-V 21.07 3.02mational preference of a given residue. Thus, theC 21.82 2.49high-occurring frequencies of Ile, Val, Leu, Phe, andY 20.28 2.00

Ala in membranes arise not only from their hydro- A 18.88 0.17phobicity but also from their high intrinsic helical T 17.92 01.08propensities in nonpolar environments. E 17.60 01.49

Furthermore, a quantitative threshold hydropho- D 16.84 02.49bicity for the spontaneous insertion of polypeptide Q 16.64 02.75

R 16.63 02.77segments into membranes was obtained through useS 16.57 02.84of this stringently controlled TM–mimetic peptideG 16.22 03.31system. Peptides inserted spontaneously from waterN 15.85 03.79into micellar membranes only when their hydropho-H 15.21 04.63bicity is equivalent to or greater than the thresholdP 14.98 04.92hydrophobicity, which can be approximated by aK 14.92 05.00

polyalanine segment of suitable length. It should benoted that peptide segments that contain residues a Estimated uncertainty in retention times, {0.01–0.05 min.

Adapted from ref. 124.of widely varying side chain polarity may still be


58 Liu and Deber

FIGURE 12 Comparison of relative hydrophobicity FIGURE 14 Experimentally determined peptide helic-determined by HPLC with different peptide models. ity (u222 nm) in LPC micelles vs mean residue hydropathyA2XA2: KKAAAXAAAAAXAAWAAXAAAKKKK- (as derived in Table VII) of peptide hydrophobic seg-NH2 (present work); AX9: Ac-EAEKAAKEXEKA- ments AAAXAAAAAXAAWAAXAAA. Single lettersAKEAEK-NH2.121

refer to the guest X residues within the correspondingpeptides. Peptides fully inserted into LPC micelles areshown as filled circles; noninserted peptides are shownas open circles. Adapted from ref. 124.erties is a highly desirable goal in molecular bioin-

formatics and biotechnology. The information de-rived from model peptides studies identifies some

acids, were synthesized by continuous-flow Fmoc solidfundamental guidelines followed by membrane pro- phase method.49 C-termini of peptides were aminatedteins, which facilitate our understanding of the gen- after cleavage from NovaSyn KR 125 resin. Purificationeral principles of membrane protein folding, and of peptides was carried out on a reverse-phase Vydac-C4lead us to approach the engineering proteins with semipreparative HPLC (10 1 250 mm, 300 A) , using adesired structures and functions. linear gradient of acetonitrile in 0.1% trifluoroacetic acid.

Purified peptides were characterized by analytical HPLC,amino acid analysis, and mass spectrometry. Concentra-tions of peptides were determined in triplicate throughMATERIALS AND METHODSquantitative amino acid analysis using Ala recovery asthe standard. Peptides were stored as solid powders atPeptide Synthesis0207C. To avoid the complexity of synthesizing a multi-ple Cys-containing peptide, for the cysteine peptide, onlyPeptides with a typical sequence of KKAAAXAAA-

AAXAAWAAXAAAKKKK-amide, where X was sub- the middle X residue was substituted by Cys, and theother two X residues were replaced by Leu. Amino acidsstituted by each of the 20 commonly occurring amino

FIGURE 13 Rank order of hydrophobicity of amino acids derived by various methods.HPLC Å hydropathy scale described in the present work. K-D Å Kyte-Doolittle hydropathyscale. 69 GES Å Goldman–Engelman–Steitz hydropathy scale.68



FIGURE 15 Distributions of mean residue hydropathy of transmembrane a-helices fromsingle-spanning integral membrane proteins (n Å 876), TM a-helices from multiple-spanningmembrane proteins (n Å 4568), and nontransmembrane a-helices of length ¢19 residues (nÅ 339). (a) Hydropathy values obtained using the scale given in Table VII, with thresholdhydropathy indicated as a solid vertical line at the value of 0.4 as calculated for the X Å Alapeptide; (b) hydropathy values obtained using the Kyte-Doolittle scale, with threshold hydropa-thy indicated as the value of 1.7 as calculated for the X Å Ala peptide. Adapted from ref. 124.

are abbreviated by one-letter symbols: A—Ala; C—Cys; Lipids, Inc. (Alabaster, AL). Lipids can be stored as pow-D—Asp; E—Glu; F—Phe; G—Gly; H—His; I—Ile; der at 0207C for 12 months. The aqueous buffer was pre-K—Lys; L—Leu; M—Met; N—Asn; P—Pro; Q— pared from 10 mM Tris HCl, 10 mM NaCl, pH 7.0. LipidGln; R—Arg; S—Ser; T—Thr; V—Val; W—Trp; micelle concentration: 10 mM lyso-phospholipids in 10Y—Tyr. mM Tris HCl, 10 mM NaCl, pH 7.0. Freshly prepared

samples were used to minimize lipid oxidation. Peptidesremained soluble and monomeric in both aqueous and

Spectroscopic Measurements membrane-mimetic media over a wide concentration range(5–250 mM), based on CD measurements and size exclu-CD measurements were performed on a Jasco-720 spectro-sion HPLC chromatography.polarimeter using a 1-mm path-length quartz cell at 257C.

Fluorescence spectra were recorded on an Hitachi F-Each spectrum was the average of four scans with buffer400 fluorescence spectrophotometer with emission andbackground subtracted. Peptide concentration was typicallyexcitation slit widths of 5 nm. Excitation wavelength was30 mM in aqueous buffer, in various lipid micellar solutions.

Synthetic LPC (C14 : 0) was purchased from Avanti Polar 280 nm. Emission spectra were recorded from 300 to 400


60 Liu and Deber

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guidelines for membrane protein engineering derived from de novo designed model peptides

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