In Vitro Selection of Membrane-spanning Leucine Zipper Protein-Protein Interaction Motifs Using POSSYCCAT

Abstract

A membrane-spanning heptad repeat motif mediates interaction between transmembrane segments. This motif was randomized with three different sets of mostly hydrophobic residues in the context of POSSYCCAT, a modified ToxR transcription activator system. The resulting combinatorial libraries were subjected to different levels of selective pressure to obtain groups of transmembrane segments that are distinguished by their ability to self-interact in bacterial membranes. Upon relating self-interaction to amino acid composition, the following conclusions were made. First, randomization with only Leu, Ile, Val, Met, and Phe resulted in unexpected robust self-interaction with little sequence specificity. Second, with more complex amino acid mixtures that represent natural transmembrane segments more closely, self-interaction critically depended on amino acid composition of the interface. Whereas the contents of Ile and Leu residues increased with the ability to self-interact, the contents of Pro and Arg residues decreased. Third, heptad repeat motifs composed of Leu, Ile, Val, Met, and Phe were ∼40-fold over-represented in transmembrane segments of single-span membrane proteins as compared with motifs composed of the more complex amino acid mixtures. This suggests that heptad motifs composed of the smaller subset of amino acids were enriched in the course of natural single-span membrane protein evolution. A membrane-spanning heptad repeat motif mediates interaction between transmembrane segments. This motif was randomized with three different sets of mostly hydrophobic residues in the context of POSSYCCAT, a modified ToxR transcription activator system. The resulting combinatorial libraries were subjected to different levels of selective pressure to obtain groups of transmembrane segments that are distinguished by their ability to self-interact in bacterial membranes. Upon relating self-interaction to amino acid composition, the following conclusions were made. First, randomization with only Leu, Ile, Val, Met, and Phe resulted in unexpected robust self-interaction with little sequence specificity. Second, with more complex amino acid mixtures that represent natural transmembrane segments more closely, self-interaction critically depended on amino acid composition of the interface. Whereas the contents of Ile and Leu residues increased with the ability to self-interact, the contents of Pro and Arg residues decreased. Third, heptad repeat motifs composed of Leu, Ile, Val, Met, and Phe were ∼40-fold over-represented in transmembrane segments of single-span membrane proteins as compared with motifs composed of the more complex amino acid mixtures. This suggests that heptad motifs composed of the smaller subset of amino acids were enriched in the course of natural single-span membrane protein evolution. positive selection system based on chromosomally integrated CAT (a modified version of the ToxR system previously used to study interactions between transmembrane segments) heptad repeat motif of Leu on an Ala host sequence chloramphenicol acetyltransferase ToxR protein lacking a transmembrane segment maltose binding protein Miller units polymerase chain reaction transmembrane segment Sequence-specific interactions between α-helical transmembrane segments are known to drive homo- and heterotypic assembly of many integral membrane proteins (1Lemmon M.A. Engelman D.M. Q. Rev. Biophys. 1994; 27: 157-218Crossref PubMed Scopus (177) Google Scholar, 2Popot J.-L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (529) Google Scholar). Self-assembly of transmembrane segment helices depends on steric complementarity of their characteristically shaped surfaces allowing for multiple van der Waals interactions (1Lemmon M.A. Engelman D.M. Q. Rev. Biophys. 1994; 27: 157-218Crossref PubMed Scopus (177) Google Scholar, 3Dieckmann G.R. DeGrado W.F. Curr. Opin. Struct. Biol. 1997; 7: 486-494Crossref PubMed Scopus (49) Google Scholar, 4Bowie J.U. Nat. Struct. Biol. 2000; 7: 91-94Crossref PubMed Scopus (33) Google Scholar). In addition, hydrogen bonding between membrane-embedded polar residues has been shown to drive interaction of model transmembrane segments (5Zhou F.X. Cocco M.J. Russ W.P. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 2000; 7: 154-160Crossref PubMed Scopus (353) Google Scholar, 6Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (341) Google Scholar, 7Zhou F.X. Merianos H.J. Brünger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (314) Google Scholar). Depending on the geometry of amino acid packing, pairs of transmembrane segment helices favor either positive or negative crossing angles (8Langosch D. Heringa J. Proteins Struct. Funct. Genet. 1998; 31: 150-160Crossref PubMed Scopus (119) Google Scholar, 9Bowie J.U. J. Mol. Biol. 1997; 272: 780-789Crossref PubMed Scopus (278) Google Scholar). Previously, we demonstrated that the interfaces of those pairs of transmembrane segments adopting positive crossing angles within bacteriorhodopsin, the photosynthetic reaction center, and cytochrome-coxidase are described by a repeated heptad (a..de.g) pattern of amino acids that is characteristic of soluble leucine zipper interaction domains (8Langosch D. Heringa J. Proteins Struct. Funct. Genet. 1998; 31: 150-160Crossref PubMed Scopus (119) Google Scholar). Apart from these multispan proteins, membrane-spanning leucine zipper domains were also found to drive assembly of certain single-span membrane proteins (10Arkin I.T. Adams P.D. MacKenzie K.R. Lemmon M.A. Brünger A.T. Engelman D.M. EMBO J. 1994; 13: 4757-4764Crossref PubMed Scopus (173) Google Scholar, 11Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 12Pinto L.H. Dieckmann G.R. Gandhi C.S. Papworth C.G. Braman J. Shaugnessy M.A. Lear J.D. Lamb R.A. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11301-11306Crossref PubMed Scopus (317) Google Scholar). Furthermore, a ga..de.ga..de.ga heptad repeat motif of Leu residues drives self-assembly of artificial transmembrane segments in natural membranes and in detergent solution (13Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Interestingly, variants of this Leu-rich heptad motif were found within the transmembrane segments of a diverse set of single-span natural membrane proteins where they appear to be important for oligomeric assembly and function (13Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 14Kubatzky K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmüller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 15Huber O. Kemmler R. Langosch D. J. Cell Sci. 1999; 112: 4415-4423Crossref PubMed Google Scholar). Here, we investigated the relationship between self-interaction of a membrane-spanning heptad motif and its amino acid composition. To this end, we randomized the ga..de.ga..de.ga motif with three different sets of mostly hydrophobic residues and expressed the resulting sequences in the context of POSSYCCAT1(Positive Selection System based onChromosomally integrated CAT), which is a modified version of the ToxR system previously used to study interactions between transmembrane segments (16Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (217) Google Scholar, 17Brosig B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (192) Google Scholar). ToxR chimeric proteins harboring the random transmembrane segment were then isolated with or without selective pressure. Subsequently, the relationship between self-assembly and amino acid composition of the interfacial positions was established. The ToxR/MalE coding region from vector pToxRΔTM (13Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) was amplified using a sense primer with an XbaI site, the ribosome binding site from pASK111 (18Skerra A. Gene. 1994; 151: 131-135Crossref PubMed Scopus (302) Google Scholar), and an antisense primer, including a sequence encoding the myc antibody tag and anXhoI site. The resulting PCR fragment was inserted into pASK111 cut with XbaI/XhoI. Upon digesting this vector with XbaI/AseI, the fragment harboring the ToxR/MalE coding region, the F1 origin, and the chloramphenicol resistance gene was isolated. This fragment was ligated to a PCR fragment of vector pBAD (Invitrogen) harboring the araBADpromoter, the araC gene, and the ColE1 origin as well as terminal XbaI and AseI sites. In the resulting vector the BamHI site of the araBAD promoter was eliminated by point mutagenesis. Upon digesting this vector withPstI/AseI, the fragment harboring thearaBAD promoter, the ToxR/MalE coding region, and thearaC gene was isolated. This fragment was ligated to a PCR fragment harboring the kanamycin resistance gene and the F1 origin, which was amplified from vector pET24d (Novagen) using a sense primer with the T2 transcription terminator from the cat2 gene fromStaphylococcus aureus plus an NdeI site and an antisense primer with a PstI site. This resulted in vector pToxRIVΔTM. Derivatives of pToxRIV encoding AZ2, L1/6/8/13/15A, or L2/5/9/12/16A transmembrane segments were made by ligating synthetic oligonucleotide cassettes encoding the desired sequences into pToxRIV previously cut with NheI and BamHI. The ctx promoter region of plasmid pLDR10-ctxΔsiglacZ (kindly supplied by H.-J. Fritz, Göttingen) was amplified using a sense primer with anXbaI site and an antisense primer where 34 nucleotides at the 3′-end were homologous to the 5′-end of the cat gene. The cat gene of plasmid pLDR10-ctxΔsiglacZ was amplified using the foregoing PCR fragment as the sense primer and an antisense primer, including the T2 terminator and a BamHI site. The resulting PCR fragment was ligated to a restriction fragment, which resulted from digesting pLDR10 W. PubMed Scopus (119) Google Scholar) and the site, a origin, and The resulting vector is The gene in vector was integrated into the site as described previously W. PubMed Scopus (119) Google Scholar). The resulting is libraries were by using a from vector and the following antisense and and to mixtures of and libraries were by the into libraries were by digesting plasmid isolated from the libraries with units of BamHI for and with units of for at This To for ToxR chimeric proteins integrated into the the were into and for at in maltose and kanamycin as described previously B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (192) Google Scholar). isolated from or from libraries were into and at in plus The was at a of into and the were to and plus or Upon for at were and were isolated and on the sequences from libraries without chloramphenicol we found that their amino acid the by and in A were for at in the of and of was as described previously (16Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (217) Google Scholar) and is in Miller units was as described with an the of the (16Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (217) Google Scholar). The transmembrane segments of single-span or multispan membrane proteins in version of were with repeat motif where to amino acid used for transmembrane segment randomization of libraries or to amino acid the residues and single-span membrane proteins, a subset of proteins that transmembrane segments was multispan membrane proteins, a subset of transmembrane segments within proteins was of the transmembrane segment is and on the we residues at either in the were the were to the membrane-spanning were using the of the sequence made by the ToxR proteins of the ToxR a transmembrane segment of and a maltose binding protein H. B. B. Fritz H.-J. EMBO J. PubMed Scopus Google Scholar). within the membrane of these ToxR are to in on the of the transmembrane segments (16Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (217) Google Scholar, 17Brosig B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (192) Google Scholar). The ToxR of the the promoter of a gene in is of ToxR assembly in the self-assembly of transmembrane segments is to we the pToxRIV vector allowing for To this end, we the ToxR promoter H. C. Fritz H.J. Biol. Chem. 1994; PubMed Scopus Google Scholar) by the araBAD promoter, which is by and by R. B. J. Mol. Biol. 1996; PubMed Scopus Google Scholar). In an we compared by different which in their to we compared the which is a previously heptad repeat motif of Leu residues (13Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), where Leu and or at and was by Ala and a ToxR protein lacking the transmembrane segment increased the in by the different decreased. between transmembrane segments of different is by the levels of ToxR protein In selection of transmembrane segments of combinatorial libraries to selective pressure where only ToxR proteins To a system allowing for positive we the gene encoding chloramphenicol acetyltransferase of the ctx A of sequence was integrated into the site of the the ToxR proteins chloramphenicol resistance to this system is segments of different be distinguished from using as an of by or we found that the of plus between and the described Upon at only these are to for transmembrane segments. a different Russ and Engelman W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) previously the system where the sequence is on a we found that harboring a between the of chloramphenicol resistance by the its and the of only of the gene, as in POSSYCCAT, increased the of the selection system to between interactions of different and positions of the ga..de.ga..de.ga motif the of pairs of transmembrane segments with positive crossing angles (8Langosch D. Heringa J. Proteins Struct. Funct. Genet. 1998; 31: 150-160Crossref PubMed Scopus (119) Google Scholar, R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) were different sets of hydrophobic amino acids by were residues to or and of the amino acids in transmembrane segments I.T. Brünger A.T. Biophys. 1998; PubMed Scopus Google Scholar, A. Engelman D.M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). Ala was for the and an sequence self-interact (13Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The sequences were inserted into the vector by mutagenesis. To the of the random transmembrane segment sequences in these libraries to self-interact, we of the the by libraries and libraries and increased selective of in these libraries Upon of This of was compared with the of ToxR proteins of membrane The this was to obtain a of the of transmembrane segments in To this end, the libraries were into and from is an that on with maltose as the only only of ToxR proteins that their the B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (192) Google Scholar, H. J. Biochem. PubMed Scopus Google Scholar). To the of this with or (13Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) were on and at a of Subsequently, this of was on and were that of to This that of proteins were eliminated by this of increased to of a a that the of random transmembrane segment those by libraries and are of membrane only the version of a of ToxR In the we the ability of the different libraries to chloramphenicol resistance to libraries were into and from to Upon the of were In with the different the libraries different to selective pressure. the of only with chloramphenicol A was for with and is of In to the of libraries and more and as a function of chloramphenicol the of of libraries and was to and of the that were This suggests a of in libraries and in with only Leu, Ile, Val, Met, and Phe to well the of the amino acid used for randomization the of transmembrane segments. This is by as well as by of chloramphenicol To obtain transmembrane segments with different we isolated ToxR proteins from with or without selective pressure. In the we isolated ToxR without selective to obtain proteins of membrane were then into to the by the ToxR to this of the sequences were into different groups or ToxR proteins from these to or that of the which to the mostly transmembrane segments were isolated without selection from libraries and as sequences to of in repeat sequences are represent Ala heptad repeat sequences that were selection by sequences were without in a repeat sequences are represent Ala heptad repeat sequences that were selection by sequences were without In the we selective pressure to obtain transmembrane segments. To this end, were selection by chloramphenicol from to the of self-interaction were then by in be ToxR proteins, as by the chloramphenicol In to sequences or were from libraries and in the of on the the of the of a set of ToxR proteins by that and sequences were expressed at levels the different appear to from different levels of ToxR the amino acid of the sequences were to they were to the of those the of the the a of residues the Ile was enriched in and and to be in The of and sequence composition were with Here, and of Ile and Leu, were in and Pro and Arg residues were to in and sequences that interactions between transmembrane segments are by the Ile and Leu and by the Pro and the Arg the sequences from the in amino acid composition and we the different were enriched at positions in the heptad shown in the Pro residues were found in positions and of sequences in This is in with Pro be to the α-helical at the or of a transmembrane for A of this study was that sequences from or This that heptad motifs composed of only Leu, Ile, Val, and residues self-interact in a the amino acid are or in natural transmembrane segments to the more complex used for libraries or To this end, we libraries the transmembrane segments of membrane proteins by with the ga..de.ga..de.ga motif of the amino acid single-span membrane protein transmembrane segments were that the of and which is the of this found that or transmembrane segments of single-span membrane proteins heptad motifs with amino acid libraries or these were in to the combinatorial and for the of these amino acids in transmembrane segments to heptad motifs Leu, Ile, Val, and were found to be over-represented ∼40-fold to those of libraries and the appear to be enriched in the course of single-span membrane protein evolution. we the of depends on the of amino acid transmembrane segment with heptad composed of mixtures of hydrophobic amino mixtures are to transmembrane the of the residues Ile, is and the Pro is in of with of Met, Met, or we only or natural transmembrane segments these for the natural to of or of In addition, we multispan membrane proteins transmembrane segments in a mostly heterotypic and Here, the were over-represented only that of these is more with single-span with multispan membrane proteins of the heptad motifs characteristic of the different libraries in natural single-span and multispan membrane of the libraries where is the of and is the of randomized positions to the of single-span membrane protein of that a motif to the to the of multispan membrane protein in that a motif to the by the of by the the were for the different of the amino acids in single-span membrane protein by the of Met, Ile, Leu, Val, and to the of the of by the of by the the were for the different of the amino acids in multispan membrane protein by the of Met, Ile, Leu, Val, and to the of the of following single-span membrane proteins the of of the libraries where is the of and is the of randomized positions to the of single-span membrane protein of that a motif to the to the of multispan membrane protein in that a motif to the by the of by the the were for the different of the amino acids in single-span membrane protein by the of Met, Ile, Leu, Val, and to the of the of by the of by the the were for the different of the amino acids in multispan membrane protein by the of Met, Ile, Leu, Val, and to the of the of in a The following single-span membrane proteins the of be that the of transmembrane segments by with the different amino acid of libraries and to and of transmembrane segments from single-span membrane proteins by multispan proteins, the are and the amino acid of libraries and that to a smaller of transmembrane segments are more of natural transmembrane segments the The heptad repeat pattern randomized was within the of pairs of transmembrane segments that positive crossing angles in membrane proteins (8Langosch D. Heringa J. Proteins Struct. Funct. Genet. 1998; 31: 150-160Crossref PubMed Scopus (119) Google Scholar). that the transmembrane segments isolated positive crossing crossing angles in soluble and membrane-spanning proteins from (8Langosch D. Heringa J. Proteins Struct. Funct. Genet. 1998; 31: 150-160Crossref PubMed Scopus (119) Google Scholar) were at the interfacial a and of into by of the in a we that interaction of the membrane-spanning domains isolated in this study is based on of the randomized amino the Ala residues are to to on the of ToxR is that were between α-helical transmembrane segments are to be based on multiple van der Waals between surfaces (1Lemmon M.A. Engelman D.M. Q. Rev. Biophys. 1994; 27: 157-218Crossref PubMed Scopus (177) Google Scholar, 2Popot J.-L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (529) Google Scholar, 3Dieckmann G.R. DeGrado W.F. Curr. Opin. Struct. Biol. 1997; 7: 486-494Crossref PubMed Scopus (49) Google Scholar, 4Bowie J.U. Nat. Struct. Biol. 2000; 7: 91-94Crossref PubMed Scopus (33) Google Scholar). Previously, residues with a to hydrogen and also been to interactions of transmembrane segments (5Zhou F.X. Cocco M.J. Russ W.P. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 2000; 7: 154-160Crossref PubMed Scopus (353) Google Scholar, 6Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (341) Google Scholar, 7Zhou F.X. Merianos H.J. Brünger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (314) Google Scholar). the amino acid for randomization in the study of these hydrogen self-interaction of the heptad pattern is to van der Waals to the of the ToxR transmembrane segments isolated in a of the libraries and their to selective we that the of the amino acid used for randomization the of transmembrane segments. A set of only hydrophobic heptad that are of self-interaction in a the of hydrophobic amino acids to in at the interfaces of the transmembrane segments that was of the In to libraries and transmembrane segments. The heptad of the were by sequence Ile and Leu residues were enriched in appear to the interaction of transmembrane segments. A of by heptad repeat of Leu, Ile, or of mixtures of that these residues a to interaction of transmembrane segments. Gurezka and D. The Pro of transmembrane segments isolated from was to to its Pro into J. Mol. Biol. PubMed Scopus Google Scholar) and has been shown to interactions between transmembrane segments O. Kemmler R. Langosch D. J. Cell Sci. 1999; 112: 4415-4423Crossref PubMed Google Scholar). Interestingly, the Pro residues of sequences were at the of the transmembrane segments where they be to the α-helical at the the Pro is in and of transmembrane segments from Pro has also been found in transmembrane segments of certain membrane protein J.-L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (529) Google Scholar), the of Pro to to on sequence Russ and Engelman W.P. Engelman D.M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar) previously randomized a sequence motif that interaction of A transmembrane which a negative (16Langosch D.L. Brosig B. Kolmar H. Fritz H.-J. J. Mol. Biol. 1996; 263: 525-530Crossref PubMed Scopus (217) Google Scholar, M.A. J.M. J. Engelman D.M. 31: PubMed Scopus Google K.R. Engelman D.M. 1997; PubMed Scopus Google Scholar). The of transmembrane segments in that study a motif W.P. Engelman D.M. J. Mol. Biol. 2000; PubMed Scopus Google Scholar), sequence the self-interaction of A transmembrane segments B. Langosch D. Protein Sci. 1998; 7: 1052-1056Crossref PubMed Scopus (192) Google Scholar, Lemmon M.A. Protein Sci. 1996; PubMed Scopus Google Scholar). In to only three of of heptad sequences and a This the that transmembrane segments positive crossing shown the of transmembrane segments from or natural transmembrane segments a heptad repeat pattern composed of Leu, Ile, Val, and are to self-interact with that of sequences from is of of its and that self-interaction of the heptad pattern is of the residues at and the transmembrane segments of single-span membrane proteins in with that a of natural transmembrane segments. of these sequences at Pro at positions their interaction by their robust and we the of amino acid was enriched in the course of membrane protein to the of libraries and found that the are over-represented ∼40-fold in single-span only in multispan membrane This suggests that the of these an and that this is with single-span with multispan This between of proteins is the transmembrane segments of the only in a and interactions within the are mostly heterotypic and in also be and in Interestingly, a of the single-span membrane proteins a pattern a of different membrane protein where interactions of transmembrane segments previously been sequences to from different are function within the which interactions between and domains Curr. Opin. 1998; PubMed Scopus Google Scholar). In addition, the function of is by self-interaction of its transmembrane segment that the to O. Kemmler R. Langosch D. J. Cell Sci. 1999; 112: 4415-4423Crossref PubMed Google Scholar). Apart from and and the interactions of transmembrane segments assembly and function of a of The also three and three of the from different previously that self-assembly of the transmembrane segment by point the of and resulted in that is for and K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmüller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In of that the as a O. D.L. 1999; PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar), we that of the transmembrane segments a for K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmüller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). between transmembrane segments also been for of and function of the of of leucine residues that within a heptad repeat pattern within its transmembrane segment of this protein and its ability to membrane PubMed Scopus Google Scholar). In with this from different that assembly of their transmembrane segments is a of these In assembly function of at three membrane protein is by self-interaction of transmembrane segments that heptad repeat characteristic of that transmembrane segment interactions are also for and function of many single-span membrane proteins transmembrane segments this of heptad repeat for with for the of in transmembrane and W. B. for