I:Forces involved membrane

in the assembly

and stabilization

of

#{149}1

proteins

W. A. CRAMER,.2

D. M. ENGELMAN,1

G. VON

HEIJNE,*

AND

D. C. REES5

‘Department

of Biological Sciences, Purdue University West Lafayette, Indiana 47907, USA; tDepartment of Molecular Biophysics and Biochemistry, Yale Univessity, New Haven, Connecticut 06611, USA; 1Department of Molecular Biology, Karolinska Institute, Center for Structural Biochemistry, NOVUM, S-14157 Huddinge, Sweden; and SDivaijon of Chemistry and Chemical Engineering, 147-75CH, California Institute of Technology, Pasadena, California 91125, USA

ABSTRACT Hydrophobic organization: Determination of the structure of the bacterial photosynthetic reaction center, bacterial porins, and bacteriorhodopsin allows a comparison of the basic structural features of integral membrane proteins. Structure parameters of membraneand water-soluble proteins are surprisingly similar, given the different dielectric environments, except for the polarity of residues on the protein surface. Hydrophobic and electrostatic forces: 1) Intramembrane helix-helix interactions that are sensitive to small structure changes can dictate assembly of membrane proteins, as indicated by reconstitution of bacteriorhodopsin from proteolytic fragments and specific dimer formation of the human erythrocyte sialoglycoprotein glycophorin A. 2) Electrostatic interactions have an important role in determining the trans-membrane orientation of integral membrane proteins of the bacterial inner membrane, as expressed by the “positive-inside” rule for the distribution of basic residues on the cis relative to the trans side of the membrane-spanning a-helices. The use of this charge asymmetry rule, in conjunction with a hydrophobicity algorithm for prediction of membrane-spanning domains, allows accurate prediction of the folding patterns of such polypeptides across the membrane. A role of electrostatic interactions in assembly and maintenance of the structure of oligomeric integral membrane protein complexes is also implied by the separation and extrusion from the membrane, at high pH, of the major hydrophobic subunits of the cytochrome b6f complex from the chioroplast thylakoid membrane. It is inferred that the hydrophobic helix-helix interactions between the subunits of this complex, whose function is electron transfer and proton translocation, are relatively weak compared to those in bacteriorhodopsin. Cramer, W. A., Engelman, D. M., von Heijne, G, Rees, D. C. Forces involved in the assembly and stabilization of membrane proteins. FASEBJ. 6: 3397-3402; 1992. Key Words: cytochrome b6f complex rule photosynthetic reaction center

THE GAP IN control teins

UNDERSTANDING

the folding and

protein

OF

and assembly complexes

glycophonin

THE

that

FORCES

of intrinsic

is reflected

.

positive-inside

stabilize

membrane

in many

aspects

CRYSTALLIZATION BRIEF HISTORY The

first

example

of the

high-resolution 1975

OF

analysis. mutagenesis

use

of two-dimensional

diffraction

(1), using

electron

Further data,

PROTEINS-

MEMBRANE

was that diffraction

crystals

with

of bacteriorhodopsin and

image

analysis, also utilizing led to a fairly complete

in

reconstruction

and

spectroscopic description

in 1990

of the folding of the seven a-helices of this 248 residue molecule in the membrane bilayer (2). There is only one other example, at present (mid-1992), of high-resolution data for two-dimensional crystals of a membrane protein, the light-harvesting

chlorophyll-binding

protein

associated

with

photosystem II of oxygenic photosynthesis (3). Well-diffracting three-dimensional crystals of the Escherichia coli porin and of the bacterial photosynthetic reaction center were obtained in 1983 (4) and 1984 (5), respectively. In the latter work, the structure of the reaction center in the bacterium Rhodopseudomonas vinidis was eventually solved to 2.3 A resolution, revealing

the

side

chain

positions,

thus

providing

the

first

high-

resolution structure of an integral membrane protein (6). A related structure was subsequently obtained for the reaction center of Rhodobacter sphaeroides (7, 8). The structure of gramnegative bacterial porin was also obtained, first from crystals of the protein from a photosynthetic bacterium, Rhodobacter capsulatus (9), and recently also from E. coli (10). The Structure of the porin subunit, consisting of 16 (3-strands, is different from that of the trans-membrane proteins discussed in the present work, in which the trans-membrane a-helix is the dominant structure motif. Reasonably well-diffracting ( 3 A) crystals have been obtained of other membrane proteins, but structure solutions have

not

yet

been

possible,

partly

because

of the

problem

of finding isomorphous heavy atom derivatives (11, 12). In summary, since the appearance of the first high-resolution two- and three-dimensional crystals of membrane proteins in 1975 and 1983, respectively, the structure of one protein has been fairly completely described from two-dimensional crystals, and the structures of two different proteins, the photosynthetic bacterial reaction center and bacterial porin obtained from analyses of three-dimensional crystals. In

and

proof

membrane biology.One indicator isthe small number of integral membrane proteins that have been successfullycrystallized for structure analysis. Another is the lack of understanding of the structural and dynamic details associated with the insertion and assembly of proteins into biological membranes.

1Summary of the symposium with this title held at the joint meeting of the American Society of Biological Chemistry and Molecular Biology

and

the

Biophysical

1992. 2To whom correspondence of

Biological

47907,

Sciences,

Society,

should

Purdue

Houston,

Texas,

be addressed,

University,

West

February

at: Department Lafayette,

IN

USA.

0892-6638/92/0006-3397/$o1.50. © FASEB 3397 m www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on October 19, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()

addition to the problems of protein purification, selection of detergents and precipitants, and construction of heavy atom derivatives, it is fair to say that a lack of understanding of the relative importance of hydrophobic and hydrophiic interactions in the stabilization, and crystallization, of membrane proteins is a major impediment to rapid progress in this area. A recent experimental suggestion that emphasizes the importance of hydrophilic, including electrostatic, interactions in the crystallization process is to use complexes containing bound Fab fragments of monoclonal antibodies for crystallization of membrane proteins (11).

HYDROPHOBIC ORGANIZATION MEMBRANE PROTELNS: THE REACTION CENTER

OF INTEGRAL PHOTOSYNTHETIC

molecular details of the forces governing the packing and organization of trans-membrane a-helices. The present discussion is based on the structural analysis carried out for Rb. sphaeroides (7). The following general structural features can be noted (13): Surface

area

A

C a

GAV

LIP

F YWHCMS Amino

TDNEQK

It

Acids

B

polarity

Recognition

a 0 U

GAVLIPFYWHCMSTDNEQKR

Amino Acids Figure

1. Comparison of amino acid composition of(A) helices in the membrane-spanning region of the Rb. sphaeroides reaction center and in water-soluble proteins (15), and (B) residues forming contacts between helices in the membrane-spanning region of the Rb. sphaeroides reaction center and water-soluble globular proteins. Modified from ref 13.

volume

and

sequence

conservation

When “surface”and ‘buried” residues facing the bilayerlipid and the other a-helices are distinguished, the surface residues are more apolar (14). This is in marked and obvious contrast to water-soluble proteins, in which the surface residues must be more polar. It is perhaps surprising that the average hydrophobicity of buried residues in water-soluble and membrane proteins is virtuallyidentical.Thus, “watersoluble proteins may be considered as modified membrane proteins with covalently attached polar groups. - .“ (7). The functional roles of the buried residues involved in helix-helix interactions are much more specific than those of surface residues that face the lipid, as implied by the significantly higher sequence conservation among the former in a comparison of four bacterial reaction center sequences (7). The composition of amino acid residues in the Rb. sphaeroides reaction center trans-membrane helices and at the helix-helix interfaces is shown (Fig. 1), in comparison to the distribution observed for water-soluble proteins (15). Among residues forming more than 5% of the helix-helix contacts, the reaction center exhibits a greater fractional abundance of phenylalanine, tryptophan, and alanine and a lower content of valine, relative to water-soluble proteins (13). The presence in glycophorin A in the trans-membrane helix of valine, which can participate in specific helix-helix interactions, is, however, noted below.

MEMBRANE

a

interior

Although the surface tension of hydrocarbon liquids is much smaller than that of water, suggesting that integral membrane proteins might have a less condensed interior and a larger, more irregular surface, the accessible surface area and packed volume of buried residues in the Rb. sphaeroides reaction center are similar to the values of these parameters in oligomeric water-soluble proteins of the same size (7). Residue

The three-dimensional structure of the bacterial photoreaction center (5-8) can serve as a basis for discussion of

and

PROTEIN in assembly

ASSEMBLY through

helix-helix

interactions

A central role for specific helix-helix interactions mediated by noncovalent forces between trans-membrane helical domains in the correct folding and assembly of polytopic and oligomeric membrane proteins was initially inferred from the demonstration that the polytopic bacteriorhodopsin (bR)3 molecule could be reconstituted in membranes from nonoverlapping proteolytic fragments (16). A correctly folded and functional molecule consisting of seven transmembrane helices and a bound retinal chromophore could be reconstituted from the fusion of two populations of lipid vesicles separately containing nonoverlapping two- and fivehelix trans-membrane chymotryptic fragments of bR. In a two-stage model based on the bR experiments for the folding and assembly of integral membrane proteins, 1) trans-

3Abbreviations:

FTIR, tosystem

Fourier

bR,

bacteriorhodopsin;

transform

II; SDS,

sodium

infrared; dodecyl

CD,

circular

GpA, glycophorin

dichroism;

A; PSII, pho-

sulfate.

The FASEB Journal CRAMER ET AL. 3398 Vol. 6 December 1992 Library (163.15.154.53) on October m www.fasebj.org by Kaohsiung Medical University 19, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()

a-helices are formed and 2) favorable interactions between these helices in the hydrophobic region of a lipid bilayer direct the assembly of the polypeptide or complex (17). The two-stage idea has been tested further in an experiment in which each of the first two helices was chemically synthesized and separately incorporated into membrane vesicles. Polarized Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic analysis showed that the net orientation of the helices was perpendicular to the plane of the bilayer (18). When these two populations of vesides were mixed with a third population containing a single fragment with the remaining helices and the vesicles were fused to allow the helices to interact, bR was formed when retinalwas added. X-ray diffractionwas used to demonstrate recovery of the bR structure (19). The first two helices can therefore be considered as separate independent folding domains. The human erythrocyte sialoglycoprotein glycophorin A (GpA) is another favorable system in which to study such helix-helixinteractionsbecause this 131-residuemolecule has a single trans-membrane helical domain containing no charged residues and is known to form tightly bound (i.e., sodium dodecyl sulfate stable) dimers (20). A chimera of staphylococcal nuclease with the trans-membrane region of GpA at its COOH terminus can be overexpressed in E. coli and also forms SDS-stable dimers. Helix-helix specificity is implied by the lossof the dimer in the presence of synthetic peptide mimicking the GpA trans-membrane domain (21), with concomitant formation of peptide-chimera heterodimers. Using the E. coli expression system, the residue specificity of the hydrophobic recognition in the transmembrane helicescan be probed through mutagenesis. Mutational analysis revealed that the dimerization of the GpA trans-membrane helix is highly specific. Subtle changes in the side chain structure at certain positions disrupted the association. In particular, changes of leucine-75, isoleucine-76, glycine-79, valine-80, glycine-83, valine-84, and threonine-87 to other small or nonpolar residues, in the helix extending from isoleucine-73 to isoleucine-95, frequently cause a partial or total loss of dimerization. For example, L75 - alanine, I76- alanine, G79-leucine, V80-* tryptophan, G83 -+ alanine, V84-’leucine, T87 -leucine, T87 alanine are disruptive. Thus, just adding a methyl group (e.g., G-’A, V L) can disrupt the interaction.The Fourier transform of the pattern of disruption shows that the sensitive positions occur every 3.9 residues, consistent with an interfacefor interactionof a supercoil of a-helices (Fig. 2). In contrast to other reported cases of interactions between trans-membrane helices, the set of interfacial residues in the GpA case contains no highly polar groups. membrane

The “positive-inside” membrane proteins

rule

for orientation

of Figure

Polar or electrostaticinteractionshave also been implicated as criticalin the assembly process. A principle governing the assembly of bacterial inner (cytoplasmic) membrane (22), and a large number of eukaryotic proteins (23), is that peripheral segments or loops containing an excess of positive charges do not tend to cross the membrane bilayer.This rule was originally proposed on the basis of statistical analysisof data in the literatureon experimentally determined polypeptide folding patterns and orientations (22, 23), and it has been formulated as the “cis-positive” or “positive-inside” rule. A strikingverificationof the rule was provided by the complete reversal of the orientation in the bacterial membrane of

STABILITY-ASSEMBLY

the NH2 and COOH termini and two membrane-spanning helices of the leader peptidase molecule after a transmembrane switch of its positive charge distribution by recombinant DNA techniques (24, 25). It does not seem to matter whether the positivecharge is provided by an arginine (pK -12) or lysine (pK -10.5) residue, and histidine (pK -6) can also exert an approximately equivalent effect if the cytoplasmic pH is made somewhat acidic (26). An interesting problem for physical chemical analysis of membrane protein systems is posed by the absence of a statistically defined asymmetry in the distribution of negative charges on the two exposed surfaces (22, 23), or at most a weak effect on reversal of the orientation of leader peptidase that could be created by a large excess of negatively charged aspartate residues at the NH2 terminus (26). Analysis of 24 polytopic bacterial inner membrane proteins with experimentally defined topographies and of the distributionof charged residues across the individual transmembrane helicesindicated that the positive-insiderule appliesto allparts of these proteins (27).An implication of this conclusion is that insertion into the bacterialmembrane can occur with localizedindependent domains consisting of two neighboring hydrophobic helicesand a connecting loop containing a relativelysmall number of positivelycharged argi-

OF MEMBRANE

PROTEINS

2. Model forthe dimerizationof the tmns-membrane

helix

of glycophorin A. The figure shows a model derived from molecular dynamics simulationsof the glycophorinhelixdimer (H. R. Treut-

1cm, M. A. Lemmon, D. M. Engelman, and A. T Br#{252}nger, submitted) that is supported by extensive mutational analysis (21). A chimeric construct of the glycophorin A imns-membrane region fused to staphylococcal nuclease was found to be dimeric in SDS gels, as is the native glycophorin A molecule. A number of studies have establishedthat the dimerization is due to an interaction of helicesin the nonpolar environment, and recentmutationalanalysis has shown thattheimportant residuesin the interaction are L75, 176, G79, V80, G83, V84, and T87. Small changes in the side chains at thesepositionsoftenresultin partialor complete lossof dimerization on gels (see text).

3399

m www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on October 19, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()

nine and lysine residues.Such an independent helicalhairpin insertion model (28) contrasts with a linear N- to C-insertion model, which may betterdescribe cotranslationalmembrane protein assembly in the endoplasmic reticulum membrane (29, 30). In the latter case, the NH2-terminal tnansmembrane segment with a favorable asymmetric charge distribution inserts into the membrane and dictates the subsequent insertion of the downstream trans-membrane segments. With the foregoing precedents, it was proposed that the positive-inside rule can be used to improve the accuracy of prediction of the folding pattern and orientation of bacterial inner membrane proteins.The following procedure was employed to describe the folding and orientation of putative polytopic membrane proteins. It predicted the correct topography for 23 of 24 bacterial membrane proteins whose topographies had previously been determined, crystallographically, biochemically, or genetically (27):

A V

Posjtjon

1. Hydrophobicity analysis was carried out to identify membrane-embedded segments, using a trapezoid sliding window for averaging that was composed of an 11-residue rectangular section and two flanking sectionseach 5 residues long. 2. The data base of 24 inner membrane proteins was used to catalog “certain” and “tentative” trans-membrane segments, whose average hydrophobicity value exceeded a certain threshold or fell in a membrane-like interval below

(a)

#{149}1 =

9

=

17

it.

3. The trans-membrane difference of positively charged groups (arginines,lysines,and the NH3 terminus) was calculated for all possible folding patterns (i.e., with certain helices, with or without tentative helices, and with the NH3 terminus on one side or the other). This protocol predicts an orientation and number of transmembrane helicesthat agree with experimental data for 23 of the 24 bacterialmembrane proteins that were tested (27). Application of this protocol to the LacY (permease) protein that has 11 certain plus 1 tentative trans-membrane helices is shown (Fig. 3).

0 .

destabilization of an oligomeric protein complex the experimental and extrinsic membrane proteins

intrinsic definition

Studies of the erythrocyte membrane system led to the view that the ability of extreme alkaline pH or high ionic strength to remove peripheral proteins from membranes leads to a clear experimental discrimination between 1) extrinsicperipheral and 2) intrinsic membrane proteins (31). This traditional approach was recently used in a study of the membrane topography of the chloroplast thylakoid membrane oligomeric cytochrome b5f complex. This complex is a centralelectron transfercomplex, present in membranes of oxygenic photosynthesis, and is related in evolution to the cytochrome bc1 complex of the mitochondrial respiratory chain and of photosynthetic bacteria (32). Exposure of the thylakoid membranes to alkaline pH was used to determine whether the polypeptide subunit of the bf complex containing the high-potential iron-sulfur (2Fe-2S) center was integral or peripheral (33). At pH values (ca. 10.5-11.5) that are not extreme for the traditional separation of peripheral and integral proteins, lateral separation in the membrane and segregation from the bcomplex were observed for its three most hydrophobic polypeptides: the 214-residue cytochrome b6 that possesses four trans-membrane helices (34), subunit iv of the complex (three helices), and the (2Fe-2S) polypeptide

.C)I

-

-

-

-

-

LII

11 (b)

Figure 3. A) Hydrophobicity plot for the LacY (permease) protein, with upper and lower thresholds defined. A tentative transmembrane

Electrostatic membrane of integral

-

0

segment

with

a mean

hydrophobicity

between

the two

thresholdsismarked with an arrow.B) Two possibletopographies for the LacY protein based on the plot in (A), with tentative transmembrane segment in black. The number of (Arg + Lys) residues at each peripheral region is shown. The experimentally correct al-

ternative

(bottom)

has a larger

positive

charge

bias.

(Rieske iron-sulfurprotein).The separation and segregation could be shown by the sequential extrusion from the membrane of these polypeptides as the pH was increased from 10.5 to 11.5(28). Of the three polypeptides, cytochrome b6, seemingly the most hydrophobic, was extruded at the least alkaline pH (10.7 for half extrusion). The most polar polypeptide in the complex, cytochrome j was not extruded. Other integral membrane proteins in the thylakoid membrane that were not extruded are the major polypeptides of the photosystem II (PSII) reaction center and the major light-harvestingchlorophyll proteins of PSII. The extrusion of the three polypeptides of the cytochrome bf complex involved release of very littlelipid and fatty acid, so in some aspects the pH-induced extrusion resembles the reversal of import. The extrusion of these three polypeptides from the membrane requires at least two electrostatic effects:1) increased electrostatic repulsion between neighboring polypeptides of the complex, documented by the ionic strength dependence

The FASEB Journal 3400 Vol. 6 December 1992 CRAMER ET AL. m www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on October 19, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber()

earlier).The weakness of such subunit interactions may be related to the energy transduction function of the cytochrome bf complex. Neutralization of the electrostaticinteractions between peripheral domains of the cytochrome bf complex would appear to be an implicit structural requirement for its proper assembly. The

research

discussed

here

was made

possible

by the following

support: U.S.PublicHealth ServiceNIH GM38323 (W.A.C.), who also thanks the Alexander von Humboldt Stiftung, together with Prof. Hartmut Michel and the Max Planck Institute for Biophysics/Frankfurt for support and hospitality during the writing of grant

this

pH

=

manuscript;

NIH

GM39546

and

NSF

1NT8612425

(D.M.E.);

the Swedish Natural Sciences Research Council and the Swedish Board for Technical Development (G.vH.); and NIH GM45162 (D.C.R.). W.A.C. thanks Prof. N. Abdulaev for helpful comments

11

and Janet this

Hollister

for dedicated

assistance

in the

preparation

of

manuscript.

REFERENCES 1. Henderson,

R., and

Unwin,

P. N. T. (1975)

Three-dimensional

model of a membrane protein obtained by electron microscopy. Nature (London) 257, 28-32 2. Henderson, R., Baldwin,J. M., Ceska, T. A., Zemlin, F., Beckmann,

E., and

Downing,

K. H. (1990)

of bacteriorhodopsin based on high microscopy. j Mol. Biol. 213, 899-929

Figure

4. Schematic diagram of the major subunits of the chloroplast cytochrome bf complex as an organized cluster (A) at neutral pH and (B) after lateral separation in the membrane bilayer at alkaline pH (ca. 11) due to electrostatic repulsion of the surface-exposed loops of the subunit polypeptides. Cytochrome b6 and subunit iv have four and three trans-membrane helices, respectively. Cytochromef and the (2Fe-2S) protein have one, and probably one, such trans-membrane helix, respectively. The negatively directed change in net charge of the polar residues in the peripheral loops is shown qualitatively, with positively (Lys, Arg) and negatively

(Asp, Glu, Tyr) charged residues circled. The changes in net electronic charge of the cytochrome b6, subunit iv, (2Fe-2S), and cytochromefpolypeptides of the complex from spinach chioroplasts (35),

upon

estimated

a pH

shift

from

7 to 11, assuming

typical

pK values,

are

to be

-10, -8, -7, and -25, respectively. The possible presence in the bf complex of an additional small hydrophobic polypeptide, the petE gene product (36), is not shown.

Model

for the structure

resolution

cryo-electron

3. Kuhlbrandt, W., and Wang, D. N. (1991) Three dimensional structure of plant light-harvesting complex determined by electron crystallography. Nature (London) 350, 130-134 4. Garavito, R. M., Jenkins, J., Jansonius, J., Karlsson, R., and Rosenbusch, J. (1983) X-ray diffraction analysis of matrix porin, an integral membrane protein from E. coli outer membranes. j Mol. Biol. 164, 313-327 5. Deisenhofer, J., Epp, 0., Miki, K., Huber, R., and Michel, H. (1984) X-ray analysis of a membrane protein complex. Electron density map at 3 A resolution and a model of the photosynthetic reaction center from Rhodopseudomonas viridis. J. Mol. Biol. 180,

385-398 6. Deisenhofer, J., and Michel, H. (1989) Nobel Lecture: The photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis. EMBO J. 8, 2149-2 169 7. Rees, D. C., Komiya, H., Yeates, T. 0., Allen, J. P.,and Feher, G. (1989) The bacterialphotosynthetic reaction center as a model for membrane proteins. Annu. Rev. Biochem. 58, 607-633 8. Chang, C.-H., El-Kabbani, 0., Tiede, D., Norris, J., and Schiffer, M. (1991) Structure of the membrane-bound protein photosynthetic reaction center from Rhodobacter sphaeroides. Biochemistry 30, 5352-5369 9. Weiss, M. S., Kreusch, A., Schiltz, E., Nestel, U., Welte, W., Weckesser, J., and Schulz, G. E. 1991) The structure of porin from Rhodobacter capsulatus at 1.8 A resolution. FEBS LetS. 280, 379-382

of polypeptide extrusion (32), would explain the phenomenon of lateralseparation of the subunit polypeptides in the membrane bilayer (Fig. 4); 2) an additional electrostatic or energetic effect is required to explain the physical extrusion from the membrane of the separated subunits. This might involve ionization of tyrosine residues in the trans-membrane helices or deprotonation of lysine residues that define the “stops” of the helices at the aqueous interfaces. The electrostatic repulsive forces at alkaline pH must then be comparable in magnitude to the attractive forces (e.g., hydrophobic helix-helixinteractions)that are required for specificassembly of polytopic oligomeric membrane protein complexes. This implies that the hydrophobic helix-helixinteractions of this complex are relatively weak compared to the strength of these interactions between the trans-membrane helices of bacteriorhodopsin or glycophorin (discussed

STABILITY-ASSEMBLY

OF MEMBRANE

PROTEINS

10. Cowan,

S. W., Schirmer,

T., Rummel,

G., Steiert,

M., Ghosh,

R., Pauptit, R. A., Jansonius, J. N., and Rosenbusch, J. (1992) Crystal structures explain functional properties of two E. coil porins. Nature (London) 358, 727-733

11. K#{252}hlbrandt,W. (1988) Three-dimensional crystallization of membrane proteins. Q Rev. Biophys. 21, 429-477 12. Michel, H. (1991) Crystallization of Membrane Proteins, CRC Press, Boca Raton, Florida 13. Rees, D. C., Chirino, A. J., Kim, K. -H., and Komiya, H. (1992) Membrane protein structure and stability: implications of the first crystallographic analyses. In Membrane Protein Structure: ExpmtJ Approaches (White, S. H., ed), Oxford University Press, New York, in press 14. Rees, D. C., DeAntonio, L., and Eisenberg, D. (1989) Hydrophobic organization of membrane proteins. Science 245, 510-513 15. Chothia, C., Levitt,M., and Richardson, D. (1981) Helix to helix packing in proteins. j Mol. BioL 145, 215-250 16. Popot, J. -L., Gerchman, S. -E., and Engelman, D. M. (1987)

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Refolding

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bilayers: a thermodyj MoL BioL 198,

J. -L., and Engelman, D. M. (1990) Membrane protein refolding and oligomerization: the two-stage model. Biochemistry 29, 4031-4037 18. Hunt, J. F.,Bousch#{233}, 0., Meyers, K. M., Rothschild,K. J., and Engelman, D. M. (1991) Biophysical studies of the integral membrane protein folding pathway. Biophys. j 59, 400a 19. Kahn, T, and Engelman, D. M. (1992) Bacteriorhodopsin can be refolded from two independently stable trans-membrane helices and the complementary five-helix fragment. Biochemistry 31, 6144-6151 17. Popot,

20. Furthmayr, H., and Marchesi, V. T. (1976) Subunit structure of human erythrocyte glycophorin A. Biochemistry 15, 1137-1144 21. Lemmon, M., Flanagan, J. M., Hunt, J. F., Adair, B. D., Bormann, B. -J., Dempsey, C. E., and Engelman, D. M. (1992) Glycophorin A dimerization is driven by specific interactions between transmembrane a-helices.j Biol. Chem. 267, 7683-7689 22. von Heijne, G. (1986) The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBOJ 5, 3021-3027 23. von Heijne, G., and Gavel, Y. (1988) Topogenic signals in integral membrane proteins. Eur. j Biochem. 174, 671-678 24. von Heijne, G. (1989) Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues.Nature (London) 341, 456-458 25. Nilsson, I., and von Heijne, G. (1990) Fine-tuning the topology of a polytopic membrane protein: role of positively and negatively charged amino acids. Cell 62, 1135-1141 26. Andersson,

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positive charged amino acids have similar effects on the topology of a polytopic transmembrane protein in E. coil. j Biol. Chem. 267, 1491-1495

3402

Vol. 6

December

1992

Heijne,G. (1992)Membrane proteinstructureprediction: hydrophobicity analysis and the “positive-inside” rule. J. MoL BioL 225, 487-494 28. Engelman, D. M., and Steitz, T A. (1981) The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell 23, 411-422 29. Hartmann, E,, Rapaport, T. A., and Lodish, H. F (1989) Predicting the orientation of eukaryoticmembrane proteins. Proc. Nati. Acad. Sd. USA 86, 5786-5790 30. High, S., Flint, N., and Dobberstein, B. (1991) Requirements for the membrane insertion of signal-anchor type proteins. j CelL BioL 113, 25-34 31. Singer, S. J. (1971) The molecular organization of biological membranes. In Structure and Function of Biological Membranes (L. I. Rothfield,ed) pp. 145-222,Academic Press,New York 32. Widger, W. R., Cramer, W. A., Herrmann, R., and Trebst,A. (1984) Sequence homology and structuralsimilaritybetween the b cytochrome of mitochondrial complex III and the chioroplastb6f complex: positionof the cytochrome b hemes in the membrane. Proc. NatI. Acad. Sci. USA 81, 674-678 33. Szczepaniak, A., Huang, D., Keenan, T W., and Cramer, W. A. (1991) Electrostatic destabilization of the cytochrome b6J complex in the thylakoid membrane. EMBOJ. 10, 2757-2764 34. Szczepaniak, A., and Cramer, W. A. (1990) Thylakoid membrane protein topography: location of the termini of the chloroplast cytochrome b6 on the stromal side of the membrane. J. Biol Chem. 265, 17720-17726 27. von

35. Widger,

W. R.,

and

Cramer,

W. A. (1991)

The

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