Cell surface expression of human cytomegalovirus (HCMV) gp55-116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response.

Cell surface expression of the human cytomegalovirus (HCMV) major envelope glycoprotein complex, gp55-116 (gB), was studied by using monoclonal antibodies and an HCMV gp55-116 (gB) recombinant vaccinia virus. HCMV-infected human fibroblasts and recombinant vaccinia virus-infected HeLa cells expresses three electrophoretically distinct proteins of Mr 170,000, 116,000, and 55,000 on their surface. These species have been previously identified within infected cells and purified virions. Two unique neutralizing epitopes were shown to be present on the cell surface gp55-116 (gB). Utilizing HeLa cells infected with the gp55-116 recombinant vaccinia virus as a specific immunosorbent, we have shown that approximately 40 to 70% of the total serum virus-neutralizing activity of a group of individuals with past HCMV infections was directed against this single envelope glycoprotein. The implications of this finding for vaccine development are discussed.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.     

The spleen focus-forming virus envelope glycoprotein is defective in oligomerization

The gp52 envelope glycoprotein of Friend spleen focus-forming virus (SFFV) is a recombinant molecule derived from Friend murine leukemia virus (MuLV) by various deletions, insertions, and substitutions. The SFFV gp52 glycoprotein, unlike MuLV envelope glycoproteins, is defective in transport to the cell surface. Only 3-5% of gp52 eventually reaches the cell surface as a processed form (gp65). Although gp52 lacks cytoplasmic tail residues found in MuLV glycoproteins, we have previously shown that this deletion is not responsible for its defective transport. In order to investigate the basis for the defective transport of gp52, we have examined the folding and assembly of gp52 molecules into oligomeric molecules. CV-1 cells infected with vaccinia virus recombinants expressing SFFV gp52 were pulse labeled and the cell extracts were fractionated by velocity centrifugation through sucrose gradients. Immediately after a 10-min pulse, gp52 was detected as a monomer in the upper part of the sucrose gradient (fractions 12 and 14) and it remained as such after a 2-h chase period. However, the processed form, gp65, was found in a lower part of the gradient (fraction 8) after a 2-h chase. The position of gp65 was found to correspond to the position of trimeric influenza hemagglutinin which was analyzed on a parallel sucrose gradient, suggesting that gp65 also exists as a trimer in this fraction. These results indicate that changes in the external domain of gp52 result in improper folding of the glycoprotein molecule, and suggest that this lack of oligomerization is responsible for the defective transport of the molecules. Only those molecules that do form oligomeric structures are transported to the Golgi complex and undergo further oligosaccharide processing, and transport to the cell surface.     

Synthesis and processing of the envelope gp55-116 complex of human cytomegalovirus.

The envelope of human cytomegalovirus has been reported to contain between three and eight glycoproteins. Major constituents of the envelope include two abundant glycoproteins with estimated molecular weights of 55,000 (gp55) and 116,000 (gp116). These two glycoproteins have been shown to exist as a disulfide-linked complex (gp55-116) within the envelope of mature virions. Utilizing a panel of monoclonal antibodies reactive with the gp55-116 complex, we characterized the synthesis and processing of these two virion proteins. Infected cells were shown to contain two glycosylated proteins of 160,000 and 150,000 daltons as well as the mature gp55 and gp116. Pulse-chase analysis indicated that gp150 was a precursor protein of gp160. The mature gp55 and gp116 were generated, in turn, by cleavage of gp160. Antigenic and structural analysis revealed that gp55 and gp116 shared little structural homology and no detectable antigenic cross-reactivity. The results of this study are discussed in relation to the synthesis of envelope proteins of other herpesviruses.     

Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB).

The human cytomegalovirus (HCMV) envelope glycoprotein complex gp55-116 was expressed in both Escherichia coli and cells infected with a recombinant vaccinia virus. E. coli produced a single protein of Mr 100,000 which approximated the size of the nonglycosylated gp55-116 precursor found in HCMV-infected cells. Cells infected with the recombinant vaccinia virus contained three intracellular forms of Mr 160,000, 150,000, and 55,000 which were detected by a monoclonal antibody reactive with gp55. Comparison of the immunological properties of these recombinant proteins indicated that several of the HCMV gp55-116 monoclonal antibodies and sera from patients infected with HCMV reacted with the vaccinia virus-derived proteins whereas a more restricted group of monoclonal antibodies recognized the E. coli-produced protein. Immunization of mice with either E. coli or vaccinia virus recombinant HCMV gp55-116 resulted in production of virus-neutralizing antibodies. In contrast to the almost exclusive production of complement-dependent neutralizing antibodies following immunization with recombinant vaccinia virus, the E. coli-derived protein induced complement-independent neutralizing antibodies.     

Processing of the gp55-116 envelope glycoprotein complex (gB) of human cytomegalovirus.

The processing pathway of the major envelope glycoprotein complex, gp55-116 (gB), of human cytomegalovirus was studied using inhibitors of glycosylation and endoglycosidases. The results of these studies indicated that the mature gp55-116 is synthesized by the addition of both simple and complex N-linked sugars to a nonglycosylated precursor of estimated Mr 105,000. In a rapid processing step, the Mr 105,000 precursor is glycosylated to a protein of Mr 150,000 (gp150) which contains only endoglycosidase H-sensitive sugar linkages. The gp150 is then processed relatively slowly to a Mr 165,000 to 170,000 species (gp165-170), which is then cleaved to yield the mature gp55-116. Monensin prevented the final processing steps of the gp150, including cleavage, suggesting that transport through the Golgi apparatus is required for complete processing. Digestion of the intracellular forms of this complex as well as the virion forms confirmed the above findings and indicated that the mature virion form of gp55 contains 8,000 daltons of N-linked sugars. The virion gp116 contains some 52,000 to 57,000 daltons of N-linked carbohydrates and approximately 5,000 daltons of O-linked sugars.     

Folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates.

Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min tag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1- to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature.     

Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein.

A detailed kinetic and quantitative analysis of the early and late biosynthetic events undergone by the human immunodeficiency virus type 1 envelope protein expressed by a recombinant vaccinia virus was performed. Early folding events that occurred in the endoplasmic reticulum included disulfide bond formation (t1/2 approximately 10 min), folding of envelope protein into a form competent to bind CD4 (t1/2 approximately 15 min), and specific and transient association and dissociation with GRP78-BiP (t1/2 approximately 25 min). After initial folding, envelope protein monomers formed noncovalently associated dimers with high efficiency (t1/2 approximately 30 min). Studies with brefeldin A, a compound that inhibits endoplasmic reticulum-to-Golgi transport, suggested that assembly occurred in the endoplasmic reticulum while cleavage of gp160 into gp120/gp41 subunits occurred in a post-endoplasmic reticulum compartment. Transport to the Golgi was monitored by modification of N-linked sugars to forms partially resistant to endoglycosidase H. The kinetics of endoglycosidase H resistance were nearly identical to the kinetics of gp160 cleavage (t1/2 approximately 80 min). Cleavage efficiency was strongly cell type dependent, ranging from 13 to 70%. By contrast, approximately 50% of the gp120 generated by the cleavage event was shed (t1/2 approximately 120 min) regardless of the cell type used. The results are discussed in terms of the overall biosynthetic pathway of the envelope protein and provide a framework with which to assess the effects of mutations on structure and function.     

The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.     

Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.     

Disulfide bonding controls the processing of retroviral envelope glycoproteins.

The mitogenic membrane glycoprotein (gp55) encoded by Friend erythroleukemia virus is inefficiently processed from the rough endoplasmic reticulum (RER) and only 3-5% reaches plasma membranes. Because this processed component (gp55P) contains larger and more complex oligosaccharides, it can be separated from RER gp55. In nonreducing conditions, gp55P is a unique disulfide-bonded dimer, whereas RER gp55 consists of monomers and dimers with diverse intrachain and interchain disulfide bonds. This suggests that gp55 folds heterogeneously and that only one homodimer is competent for export from the RER. Pulse-chase analyses of gp55 components labeled with radioactive amino acids indicated that formation of diverse disulfide-bonded components occurred within minutes of polypeptide synthesis and that malfolded components did not later isomerize to generate dimers competent for export from the RER. Chemical studies suggested that all 12 cysteines of gp55 were oxidized within 5 min after synthesis of the protein. In contrast, the envelope glycoprotein precursor (gPr90) encoded by a replication-competent murine leukemia virus folds more homogeneously, and it is then processed and cleaved to form an extracellular glycoprotein gp70 plus a transmembrane protein p15E. The fully processed glycoprotein contains an unoxidized cysteine sulfhydryl that isomerizes reversibly with a disulfide bond that links gp70 to p15E. Consequently, only a proportion of gp70 and p15E is disulfide-bonded, and dissociation occurs when the environment becomes even slightly reducing. The gp55 glycoprotein appears to be an extreme example of protein malfolding associated with imprecise and irreversible disulfide bonding. We discuss evidence that folding inefficiencies are common for retroviral proteins that have newly evolving pathogenic functions.     

Differential recognition of epitopes present on monomeric and oligomeric forms of gp160 glycoprotein of human immunodeficiency virus type 1 by human monoclonal antibodies.

The mechanism of infectivity neutralization of human immunodeficiency virus type 1 (HIV-1) by Ig is poorly understood. Three human monoclonal antibodies (mAbs 1b12, 2G12 and 2F5) that are able to neutralize primary isolates of HIV-1 in vitro have been shown to act synergistically. In the present study this synergy was analyzed by measuring the epitope accessibility and binding kinetics for these three mAbs with respect to monomeric and oligomeric env protein gp160 IIIB using surface plasmon resonance. The results indicate that oligomerization of gp160 affects the accessibility of some of the epitopes recognized by the mAbs and provide some insight into the mechanism of synergy between different anti-(HIV-1) mAbs.     

The oligomeric T4 primase is the functional form during replication

Replisome DNA primases are responsible for the synthesis of short RNA primers required for the initiation of repetitive Okazaki fragment synthesis on the lagging strand during DNA replication. In bacteriophage T4, the primase (gp61) interacts with the helicase (gp41) to form the primosome complex, an interaction that greatly stimulates the priming activity of gp61. Because gp41 is hexameric, a question arises as to whether gp61 also forms a hexameric structure during replication. Several results from this study support such a structure. Titration of the primase/single-stranded DNA binding followed by fluorescence anisotropy implicated a 6:1 stoichiometry. The observed rate constant, k(cat), for priming was found to increase with the primase concentration, implicating an oligomeric form of the primase as the major functional species. The generation of hetero-oligomeric populations of the hexameric primase by controlled mixing of wild type and an inactive mutant primase confirmed the oligomeric nature of the most active primase form. Mutant primases defective in either the N- or C-terminal domains and catalytically inactive could be mixed to create oligomeric primases with restored catalytic activity suggesting an active site shared between subunits. Collectively, these results provide strong evidence for the functional oligomerization of gp61. The potential roles of gp61 oligomerization during lagging strand synthesis are discussed.     

Homooligomerization of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 occurs before the acquisition of correct intramolecular disulfide bonds and mature immunoreactivity.

The posttranslational maturation of the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza type 3 virus (PIV3) was investigated in pulse-chase experiments in which folding was monitored by immunoprecipitation with conformation-dependent antibodies and gel electrophoresis under nonreducing conditions and oligomerization was monitored by chemical cross-linking and sedimentation in sucrose gradients. The acquisition of mature immunoreactivity and the formation of correct intramolecular disulfide bonds were concurrent events, with half-times of approximately 10 to 15 min. The finding that newly synthesized HN had little reactivity with postinfection cotton rat serum or with most of the members of a panel of HN-specific monoclonal antibodies indicated that the major epitopes of the PIV3 HN protein are highly conformational in nature. Chemical cross-linking studies indicated that the mature HN protein is present in homoligomers, which are probably tetramers. These findings are consistent with recent observations for the HN protein of Sendai virus (S.D. Thompson, W.G. Laver, K.G. Murti, and A. Portner, J. Virol. 62:4653--4660, 1988; S. Vidal, G. Mottet, D. Kolakofsky, and L. Roux, J. Virol. 63:892--900, 1989). Surprisingly, analysis of pulse-labeled HN protein by sedimentation on sucrose gradients after labeling periods of as little as 2 min indicated that it was present intracellularly only in oligomeric form. The same results were obtained when the labeling period was preceded by a 1.5-h cycloheximide treatment to clear the endoplasmic reticulum of presynthesized HN protein, which indicated that the oligomerization did not involve the incorporation of newly synthesized monomers into partially assembled oligomers. Subsequent chase incubations did not significantly alter the sedimentation profile or stability of the oligomeric forms, suggesting that oligomers detected after short labeling periods were tetramers. Association with cellular proteins did not appear to be responsible for the sedimentation of newly synthesized HN protein as an oligomer. The absence of a detectable monomeric form of intracellular HN protein raised the possibility that oligomerization is cotranslational, and it is possible that the type II membrane orientation of the HN protein might be an important factor in its mode of oligomerization.     

Structure and folding of bacteriophage T4 gene product 9 triggering infection. II. Study Of conformational changes of gene product 9 mutants using monoclonal antibodies

Gene product 9 (gp9) of bacteriophage T4, whose spatial structure we have recently solved to 2.3 A resolution, is a convenient model for studying the folding and oligomerization mechanisms of complex proteins. The gp9 polypeptide chain consists of 288 amino acids forming three domains. Three monomers, packed in parallel, assemble to a functionally active protein. The main aim of this work was to study conformational changes and trimerization of gp9 deletion mutants using monoclonal antibodies (mAbs). We selected a set of mAbs interacting with the amino, middle, and carboxyl regions of the protein, respectively. Eighteen mAbs bind to native as well as to denatured protein, and two mAbs bind to denatured protein only. Using mAbs, we found that deletions of the gp9 N-terminal region result in conformational changes in the middle and C-terminal domains. The study of mAb binding to the CDelta. truncated mutant by competitive ELISA and immunoblotting shows that the C-terminus of the gp9 sequence is essential for protein trimerization and stability. A single point substitution of the Gln282 residue causes formation of a labile trimer that has significant conformational changes in the protein domains. The results of our study show that folding and trimerization of gp9 is a cooperative process that involves all domains of the protein.     

Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities.

We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env.     

Steps in maturation of influenza A virus neuraminidase.

We have studied the maturation of the influenza A virus neuraminidase (NA), using monoclonal antibodies (MAbs) with different conformational specificities against the head domains of the N8 NA. The results obtained with radioimmunoprecipitation, together with previously published information, suggest the following steps in maturation of this molecule. First, the folding of the nascent NA leads to formation of the epitope recognized by MAb N8-10, a step that depends on the formation of intramolecular disulfide bonds. Second, monomers form dimers by an intermolecular disulfide linkage in the stalk, with a t1/2 of 2.5 min. Third, the epitope recognized by MAb N8-82 appears after dimerization, suggesting that oligomeric NAs may undergo conformational change with a t1/2 of 8 min. Finally, a tetramer-specific epitope recognized by MAb N8-4 appears on the NA with a t1/2 of 13 min. Epitope detection by MAb N8-4 was inhibited by tunicamycin treatment, suggesting that glycosylation of this molecule is required for proper tetramerization. Each of these proposed steps occurs in the endoplasmic reticulum of host cells, as demonstrated by treatment of virus-infected cells with brefeldin A or carbonyl cyanide m-chlorophenylhydrazine; subsequently, tetrameric NA is transported to the Golgi apparatus, where oligosaccharide processing is completed. Our findings also provide a possible explanation--lack of a functionally active conformation--for the absence of enzymatic function by NA monomers.     

The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide.

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein is composed of a soluble glycopolypeptide gp120 and a transmembrane glycopolypeptide gp41. These subunits form non-covalently linked oligomers on the surface of infected cells, virions and cells transfected with the complete env gene. Two length variants of the extracellular domain of gp41 (aa 21-166 and aa 39-166), that both lack the N-terminal fusion peptide and the C-terminal membrane anchor and cytoplasmic domain, have been expressed in insect cells to yield soluble oligomeric gp41 proteins. Oligomerization was confirmed by chemical cross-linking and gel filtration. Electron microscopy and circular dichroism measurements indicate a rod-like molecule with a high alpha-helical content and a high melting temperature (78 degrees C). The binding of monoclonal antibody Fab fragments dramatically increased the solubility of both gp41 constructs. We propose that gp41 folds into its membrane fusion-active conformation, when expressed alone.     

The tetrameric α-helical membrane protein GlpF unfolds via a dimeric folding intermediate

Many membrane proteins appear to be present and functional in higher-order oligomeric states. While few studies have analyzed the thermodynamic stability of α-helical transmembrane (TM) proteins under equilibrium conditions in the past, oligomerization of larger polytopic monomers has essentially not yet been studied. However, it is vital to study the folding of oligomeric membrane proteins to improve our understanding of the general mechanisms and pathways of TM protein folding. To investigate the folding and stability of the aquaglyceroporin GlpF from Escherichia coli, unfolding of the protein in mixed micelles was monitored by steady-state fluorescence and circular dichroism spectroscopy as well as by seminative sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. On the basis of our results, it appears most likely that GlpF unfolds in a two-step process, involving the equilibrium of tetrameric, dimeric, and monomeric GlpF species. A kinetic analysis also indicates an intermediate along the kinetic GlpF unfolding pathway, and thus, two phases are involved in GlpF unfolding. While three-state unfolding pathways and a dimeric folding intermediate are not uncommon for water-soluble proteins, a stable (un)folding intermediate with a decreased oligomeric structure has not been detected or reported for any α-helical membrane protein.