Bacteriorhodopsin can be refolded from two independently stable transmembrane helixes and the complementary five-helix fragment

Kahn, Theodore W.; Engelman, Donald M.

doi:10.1021/bi00141a027

Cited by 150 publications

(96 citation statements)

References 36 publications

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“…10 for review). The same kind of behavior has been documented in vitro by using fragments of proteins (14)(15)(16)(17). That fragments can reassociate and function argues that the covalent linkage between them, although perhaps adding stability and͞or control of expression, is not always essential.…”

Section: Methods and Resultsmentioning

confidence: 64%

Transmembrane protein domains rarely use covalent domain recombination as an evolutionary mechanism

Liu

Gerstein

Engelman

2004

Proc. Natl. Acad. Sci. U.S.A.

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Recombination of evolutionarily unrelated domains is a mechanism often used by evolution to produce variety in soluble proteins. By using a classification of polytopic transmembrane domains into families, we examined integral membrane proteins for evidence of this mechanism. Surprisingly, we found that domain recombination is not common for the transmembrane regions of membrane proteins, a majority of integral membrane proteins containing only a single transmembrane domain. We suggest that noncovalent oligomeric associations, which are common in membrane proteins, may provide an alternative source of evolutionary diversity. Membrane proteins are known to evolve diversity of function by gene duplication followed by divergent evolution, and by recombination of homologous but different subunits. However, the strategy of fusing evolutionarily unrelated transmembrane domains has not been examined for membrane proteins, even though it is commonly observed in soluble proteins. Protein domains are considered to be structural units that can be mixed to facilitate evolution (1-4), either by recombination of homologous or of nonhomologous structures. Studies of domain recombination in proteomes from archae, prokarya, and eukarya by using the structure-based classification (5) shows that a large majority of domains (Ϸ65% in prokarya and Ϸ80% in eukarya) are combined with other domains (6). This result supports the idea that recombination of domains could generate new protein structures and functions in the course of evolution. However, the result applies principally to soluble proteins, as the structural database is strongly biased in favor of them. By using our classification of polytopic membrane proteins into Ϸ650 families (7), we have been able to study domain combinations in integral membrane proteins and find that they are much less abundant than in the soluble protein cases. Methods and ResultsBased on sequence similarities and the number of putative transmembrane helices (8), we classified families of polytopic membrane domains in 26 genomes (8 in archaea, 14 in prokarya, and 4 in eukarya). A family is required to have at least four members, each with at least two putative transmembrane helices. The classification was based in part on the Pfam assignments (9) and by clustering based on sequence similarities (7). Pfam is a large collection of families based on multiple sequence alignments and profile-hidden Markov models, leading to the classification of Ͼ3,000 families (Pfam release 6.1). Most (95%) polytopic membrane domains defined in the families have relatively short loops (Ͻ75 aa) between transmembrane helices. We identified Ͼ650 families, corresponding to Ϸ61% of all predicted helical transmembrane proteins (7).Because they are the best defined, we chose to examine the cases in the manually curated Pfam-A classified families (Fig. 1A), and we found that most integral membrane proteins (Ϸ78% for archaea and prokarya and Ϸ67% for eukarya) contain only a single classified membrane domain. It follows that the lev...

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Section: Methods and Resultsmentioning

confidence: 64%

Transmembrane protein domains rarely use covalent domain recombination as an evolutionary mechanism

Liu

Gerstein

Engelman

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Transmembrane domains (TMs) are believed to be arranged in a barrel-like structure with a tightly packed core (2)(3)(4). Recent studies have suggested that GPCRs are composed of several independent folding units (5)(6)(7)(8)(9)(10)(11)(12)(13). Specifically, coexpression of polypeptides generated by splitting the muscarinic receptor in either the extracellular or the intracellular loops resulted in assembly of the molecules, which exhibited receptor-like function (6,7).…”

Section: G-protein-coupled Receptors (Gpcr)mentioning

confidence: 99%

Inhibition of G-protein-coupled Receptor Function by Disruption of Transmembrane Domain Interactions

Tarasova¹,

Rice²,

Michejda³

1999

Journal of Biological Chemistry

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“…First, the denatured apoprotein can be spontaneously refolded to the native state with quantitative recovery of secondary structure, chromophore binding, and proton-pumping activity (5,6). Reconstitution of the native structure has also been accomplished with complementary combinations of proteolytic fragments and synthetic peptides, comprising one or more of the transmembrane regions (7)(8)(9)(10)(11). Furthermore, refolding and chromophore binding has been demonstrated for numerous mutants containing amino acid substitutions, deletions, or insertions (12)(13)(14)(15).…”

Section: Bacteriorhodopsin (Br)mentioning

confidence: 99%

Refolding of Bacteriorhodopsin from Expressed Polypeptide Fragments

Marti

1998

Journal of Biological Chemistry

115

112

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Bacteriorhodopsin is a heptahelical membrane protein that can be refolded to the native state following denaturation. To analyze the in vitro folding process with independent structural domains, eight fragments comprising two (AB, FG), three (AC, EG), four (AD, DG) or five (AE, CG) of the transmembrane segments were produced by expression in Escherichia coli. The polypeptides were purified to homogeneity by solvent extraction of E. coli membranes, repeated phase separation, and anion-exchange chromatography employing the C-terminal tail of bacteriorhodopsin for adsorption. Upon reconstitution into phospholipid/detergent micelles pairs of complementary fragments (AB⅐CG, AC⅐DG, AD⅐EG, and AE⅐FG) assembled in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency. Together with previous studies, these results demonstrate that the covalent connections in each of the six interhelical loops are dispensable for a correct association of the helices. The different loops, however, contribute to the stability of the folded structure, as shown by increased susceptibilities toward denaturation in SDS and at acidic pH, and decreased Schiff base pK a values for the AB⅐CG, AC⅐DG, AD⅐EG, and AE⅐FG complexes, compared with the intact protein. Notably, the heptahelical bundle structure was also generated by all possible combinations of pairs of overlapping fragments, containing one (AC⅐CG, AD⅐DG, AE⅐EG), two (AD⅐CG, AE⅐DG), or three (AE⅐CG) redundant helices. The spectral properties of the chromophores indicate that the retinal-binding pocket of the AC⅐CG, AD⅐CG, and AE⅐CG complexes is formed by helices A and B of the respective N-terminal fragment and the C-terminal CG fragment, whereas the AD⅐DG, AE⅐DG, and AE⅐EG complexes are likely to adopt a heptahelical bundle structure analogous to AD⅐EG. The combined data show that the specificity of the helix assembly of bacteriorhodopsin is influenced by connectivities provided by the C-D and E-F surface loops. Bacteriorhodopsin (BR)1 is an integral membrane receptor that functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium (1-4). Several features make BR highly attractive for in vitro studies of membrane protein folding and assembly. First, the denatured apoprotein can be spontaneously refolded to the native state with quantitative recovery of secondary structure, chromophore binding, and proton-pumping activity (5, 6). Reconstitution of the native structure has also been accomplished with complementary combinations of proteolytic fragments and synthetic peptides, comprising one or more of the transmembrane regions (7-11). Furthermore, refolding and chromophore binding has been demonstrated for numerous mutants containing amino acid substitutions, deletions, or insertions (12-15). By using time-resolved spectroscopy, transient intermediates in the folding process of native BR have recently been identified (16, 17). Second, the structure of BR has been solved at high resolution, revealing the detai...

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Bacteriorhodopsin can be refolded from two independently stable transmembrane helixes and the complementary five-helix fragment

Cited by 150 publications

References 36 publications

Transmembrane protein domains rarely use covalent domain recombination as an evolutionary mechanism

Transmembrane protein domains rarely use covalent domain recombination as an evolutionary mechanism

Inhibition of G-protein-coupled Receptor Function by Disruption of Transmembrane Domain Interactions

Refolding of Bacteriorhodopsin from Expressed Polypeptide Fragments

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