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1.
FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein
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A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall alpha-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage. 相似文献
2.
FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association
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A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state F?rster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems. The applicability of the simulation-based fitting approach was verified on simulated FRET data and then applied to determine the structural properties of the well-known major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants Y24C, G38C, and T46C of this protein were produced and specifically labeled with the fluorescence label AEDANS. The energy transfer data from the natural tryptophan at position 26, which is used as a donor, to AEDANS were analyzed assuming a helix model for the transmembrane domain of the protein. As a result of the FRET data analysis, the topology and bilayer embedment of this domain were quantitatively characterized. The resulting tilt of the transmembrane helix of the protein is 18 +/- 2 degrees. The tryptophan is located at a distance of 8.5 +/- 0.5 A from the membrane center. No specific aggregation of the protein was found. The methodology developed here is not limited to M13 major coat protein and can be used in principle to study the bilayer embedment of any small protein with a single transmembrane domain. 相似文献
3.
New insights into the low-resolution structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process. 相似文献
4.
During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane. 相似文献
5.
Vos WL Koehorst RB Spruijt RB Hemminga MA 《The Journal of biological chemistry》2005,280(46):38522-38527
M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or F?rster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight alpha-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care. 相似文献
6.
Bashtovyy D Marsh D Hemminga MA Páli T 《Protein science : a publication of the Protein Society》2001,10(5):979-987
The family of three-dimensional molecular structures of the major coat protein from the M13 bacteriophage, which was determined in detergent micelles by NMR methods, has been analyzed by constrained geometry optimization in a phospholipid environment. A single-layer solvation shell of dioleoyl phosphatidylcholine lipids was built around the protein, after replacing single residues by cysteines with a covalently attached maleimide spin label. Both the residues substituted and the phospholipid were chosen for comparison with site-directed spin labeling EPR measurements of distance and local mobility made previously on membranous assemblies of the M13 coat protein purified from viable mutants. The main criteria for identifying promising candidate structures, out of the 300 single-residue mutant models generated for the membranous state, were 1) lack of steric conflicts with the phospholipid bilayer, 2) good match of the positions of spin-labeled residues along the membrane normal with EPR measurements, and 3) a good match between the sequence profiles of local rotational freedom and a structural restriction parameter for the spin-labeled residues obtained from the model. A single subclass of structure has been identified that best satisfies these criteria simultaneously. The model presented here is useful for the interpretation of future experimental data on membranous M13 coat protein systems. It is also a good starting point for full-scale molecular dynamics simulations and for the design of further site-specific spectroscopic experiments. 相似文献
7.
Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage 总被引:9,自引:0,他引:9
The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions. 相似文献
8.
Cysteine residues in the transmembrane regions of M13 procoat protein suggest that oligomeric coat proteins assemble onto phage progeny
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The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the -6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process. 相似文献
9.
The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection. M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies. An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments. The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei. Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics. A model is proposed for the structure of a coat protein dimer in detergent micelles. 相似文献
10.
Fractionation of membrane vesicles from coliphage M13-infected Escherichia coli. 总被引:1,自引:1,他引:0
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W Wickner 《Journal of bacteriology》1976,127(1):162-167
Membrane vesicles were prepared by osmotic lysis of spheroplasts from M13-infected Escherichia coli. Reduced nicotinamide adenine dinucleotide (NADH) oxidase (reduced NAD: oxidoreductase, EC 1.6.99.3) and Mg2+-Ca2+-activated adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3), which are normally localized to the inner surface of the cytoplasmic membrane, were 50% acceesible to their polar substrates in these vesicles. The major coat protein of coliphage M13 is also bound to the cytoplasmic membrane (prior to phage assembly) but with its antigenic sites exposed to the exterior of the cell. Antibody to M13 coat protein was used to fractionate membrane vesicles. Neither agglutinated nor unagglutinated vesicles had altered NADH oxidase and adenosine triphosphatase specific activities. This is inconsistent with such vesicles being a mixture of correctly oriented and completely inverted membrane sacs and suggests that NADH oxidase, adenosine triphosphatase, M13 coat protein, or all three proteins rearrange during vesicle preparation. 相似文献
11.
Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display. 相似文献
12.
Chen M Xie K Nouwen N Driessen AJ Dalbey RE 《The Journal of biological chemistry》2003,278(26):23295-23300
Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins. We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature. This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein. However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures. Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal. These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms. 相似文献
13.
14.
The major capsid protein of M13 bacteriophage is incorporated at each stage of infection into the host plasma membrane with its amino terminus exposed on the outer surface. Purified M13 coat protein is incorporated with the same asymmetry into synthetic phosphatidylcholine vesicles formed near the Tm of the lipid by a cholate dilution technique. We now report that the lipid in the pre-dilution mixture exists as mixed micelles of uniform size. Prior to dilution, the coat protein is present in at least two states of aggregation, both of which behave similarly in the model membrane assembly reaction. No detectable lipid-protein interaction occurs prior to dilution. Upon dilution there is rapid production of small closed vesicles and coat protein is converted to a chymotrypsin-resistant form, presumably reflecting its incorporation into these vesicle bilayers. Formation of large ( diameter) vesicles occurs slowly with preservation of coat protein asymmetry and internal volume. A model for this assembly reaction is proposed. 相似文献
15.
C M Deber Z Li C Joensson M Glibowicka G Y Xu 《The Journal of biological chemistry》1992,267(8):5296-5300
Although transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, beta-sheet promoters (Val, Ile, Thr) often account for approximately 40% of TM residue composition. We are examining the conformational role(s) of these residues, using as a model system the major coat protein of the filamentous bacteriophage M13. This 50-residue protein, which is located at the Escherichia coli host membrane during phage reproduction, contains a prototypic 19-residue hydrophobic midregion (residues 21-39: YIGYAWAMVVVIVGATIGI). Using "Eckstein" site-directed mutagenesis, we have generated several viable M13 coat protein mutants with beta-branched amino acid substitutions within their TM region. Mutant coat proteins, including Ile32----Val (I32V) and Ala27----Thr (A27T), were obtained in milligram quantities by growing M13 mutant phages in liter preparations, confirming that these coat proteins are capable of assuming their normal biological function(s) in phage reproduction. Circular dichroism spectroscopy performed in the membrane-mimetic medium of deoxycholate micelles indicated comparable alpha-helical contents of mutants I32V and A27T to wild-type protein. 13C nuclear magnetic resonance experiments with mutant A27T demonstrated that the combination of additional beta-branched content and introduction of an -OH substituent induced chemical shift and temperature-dependent changes and influenced the local protein environment at sites up to 12 residues remote from the mutation site. In contrast, mutant I32V (of which a salient feature is a mid-TM pentavaline segment) behaved very similarly to wild-type coat. These findings are interpreted in terms of the range of TM secondary structure and stability which can be accommodated by viable M13 coat protein mutants. 相似文献
16.
The major coat protein of bacteriophage M13 has been reconstituted into phospholipids with a composition comparable to that found in the host (Escherichia coli) inner membrane. Reconstitution experiments have revealed conditions in which the alpha-oligomeric state is favored over the beta-polymeric state. Discrimination between the two states of the membrane-bound coat protein (alpha-oligomeric and beta-polymeric states) has been achieved using high-performance size-exclusion chromatography and circular dichroism. Interprotein electrostatic interactions, probably induced by head-tail binding, are initiated and facilitating the aggregation-related conformational change process, in which alpha-oligomeric coat protein is converted into beta-polymeric coat protein. A model for this beta-polymerization process of the coat protein is presented. The alpha-helical protein has been studied by the in situ Trp fluorescence quantum yield. This shows that the average distances between coat proteins decrease upon lowering the L/P ratio. In situ cross-linking reactions of the coat protein at high L/P ratios reveal a monomeric state, thus excluding specific aggregation of the coat protein. A monomeric state of detergent-solubilized coat protein is also observed using SDS-PAGE and SDS-HPSEC. On the basis of these results, the smallest in situ aggregational entity of the coat protein is proposed to be a monomer. This finding is discussed in relation to the functional state of the M13 coat protein in the membrane-bound assembly and disassembly processes during infection. 相似文献
17.
Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified. 相似文献
18.
Polyoma large T antigen regulates the integration of viral DNA sequences into the genome of transformed cells 总被引:34,自引:0,他引:34
The major coat protein of coliphage M13 is an integral protein of the E. coli plasma membrane prior to its assembly into new virus particles. It is generated from its precursor, procoat, by a membrane-bound leader peptidase. We now describe the reconstitution of a highly purified preparation of this enzyme into vesicles of E. coli phospholipids. These vesicles bind procoat made in vitro and procoat isolated from in vitro synthesis. Both the crude and the purified substrates were converted posttranslationally to coat protein. A significant proportion of the coat protein becomes inserted into the vesicle bilayer, with the N terminus facing the vesicle interior and the C terminus exposed to the external medium. These results strongly suggest that highly purified leader peptidase from E. coli and phospholipids are the only components necessary to mediate the binding, processing and insertion of this integral membrane protein. 相似文献
19.
The major coat protein of M13 bacteriophage has been incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, deuterated in the trimethyl segments of the choline headgroup (DMPC-d9). Two-component deuterium and phosphorus-31 NMR spectra have been observed from bilayer complexes containing the coat protein, indicating slow exchange (on the deuterium quadrupole anisotropy and phosphorus-31 chemical shift averaging time scales) of lipid molecules of less than 10(3) Hz between two motionally distinct environments in the complexes. The fraction of the isotropic spectral component increases with increasing M13 protein concentration, and this component is attributed to lipid headgroups, which are disordered relative to their order in protein-free bilayers. The activation energy of the fast local motions of the trimethyl groups of the choline residue in the headgroup decreases from 23 kJ mol-1 in the pure lipid bilayers to 20 kJ mol-1 for the protein-associated lipid headgroups. The chemical exchange rate of lipid molecules between the two motionally distinct environments has been estimated to be 20-50 Hz by steady-state line-shape simulations of the deuterium spectra of DMPC-d9/M13 coat protein complexes using exchange-coupled modified Bloch equations. The off-rate was, as expected from one-to-one exchange, independent of the L/P ratio; tau off -1 = 0.23 kHz. It is suggested that the protein-associated lipid may be trapped between closely packed parallel aggregates of M13 coat protein and that the high local concentration of protein in a one-dimensional arrangement in lipid bilayers may be required for the fast reassembly of phage particles before release from an infected cell. 相似文献
20.
Many membrane proteins and most secreted proteins are initially made as precursors with an N-terminal leader sequence. We now report the isolation of M13 procoat, the precursor of the membrane-bound form of M13 coat protein. There are 40 000 copies of M13 procoat protein/cell during M13 amber 7 virus infection. Purified procoat is quantitatively cleaved by isolated leader peptidase to yield mature-length coat protein. Rabbit antibodies to M13 procoat will precipitate procoat but not coat, suggesting that the antibody molecules are specifically recognizing the leader sequence or the conformation which it induces in the whole procoat molecule. 相似文献