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• W2079392253 abstract "The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile12 and Val14 are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile12 on one subunit and Val14 on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport. The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile12 and Val14 are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile12 on one subunit and Val14 on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport. The twin-arginine translocation (Tat) 5The abbreviations used are: Tattwin-arginine translocationMOPS4-morpholinepropanesulfonic acidMTSL1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methylC12E9nonapolyoxyethylene dodecyl ether. transports folded proteins across the cytoplasmic membrane of bacteria and archaea and across the thylakoid membrane of plant chloroplasts (1Berks B.C. Palmer T. Sargent F. Adv. Microb. Physiol. 2003; 47: 187-254Crossref PubMed Scopus (205) Google Scholar, 2Lee P.A. Tullman-Ercek D. Georgiou G. Annu. Rev. Microbiol. 2006; 60: 373-395Crossref PubMed Scopus (244) Google Scholar, 3Cline K. Theg S.M. The Enzymes, Molecular Machines Involved in Protein Transport across Cellular Membranes. Vol. XXV. Elsevier, San Diego, CA2007: 455-485Google Scholar, 4Natale P. Brüser T. Driessen A.J. Biochim. Biophys. Acta. 2008; 1778: 1735-1756Crossref PubMed Scopus (345) Google Scholar). Proteins are targeted to the Tat pathway by amino-terminal signal peptides bearing a consensus sequence motif that includes consecutive arginine residues (5Chaddock A.M. Mant A. Karnauchov I. Brink S. Herrmann R.G. Klösgen R.B. Robinson C. EMBO J. 1995; 14: 2715-2722Crossref PubMed Scopus (226) Google Scholar, 6Berks B.C. Mol. Microbiol. 1996; 22: 393-404Crossref PubMed Scopus (561) Google Scholar). Transport through the Tat pathway is driven by the transmembrane proton electrochemical gradient (7Mould R.M. Robinson C. J. Biol. Chem. 1991; 266: 12189-12193Abstract Full Text PDF PubMed Google Scholar). The Tat pathway is vital for many cellular processes, including biogenesis of respiratory and photosynthetic electron transfer chains, formation of the bacterial cell envelope, establishing the nitrogen fixing symbiosis, and bacterial pathogenesis (8Berks B.C. Palmer T. Sargent F. Curr. Opin. Microbiol. 2005; 8: 174-181Crossref PubMed Scopus (186) Google Scholar). twin-arginine translocation 4-morpholinepropanesulfonic acid 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl nonapolyoxyethylene dodecyl ether. In the bacterium Escherichia coli the minimal components of the Tat machinery are the integral membrane proteins TatA, TatB, and TatC (9Weiner J.H. Bilous P.T. Shaw G.M. Lubitz S.P. Frost L. Thomas G.H. Cole J.A. Turner R.J. Cell. 1998; 93: 93-101Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 10Bogsch E.G. Sargent F. Stanley N.R. Berks B.C. Robinson C. Palmer T. J. Biol. Chem. 1998; 273: 18003-18006Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 11Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (443) Google Scholar, 12Sargent F. Stanley N.R. Berks B.C. Palmer T. J. Biol. Chem. 1999; 274: 36073-36082Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). TatA and TatB are sequence-related but functionally distinct proteins. TatA is predicted to be composed of an amino-terminal transmembrane helix (also termed helix-α1) separated by a short hinge region from an amphipathic helix (helix-α2), which is in turn followed by an unstructured, polar, carboxyl-terminal region. The amphipathic helix is proposed to lie along the membrane surface but may undergo a change of topology related to function in which the helix changes location to become membrane-spanning (13Gouffi K. Gérard F. Santini C.L. Wu L.F. J. Biol. Chem. 2004; 279: 11608-11615Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). TatA purified in detergent solution forms large, homo-oligomeric complexes of variable size (14Porcelli I. de Leeuw E. Wallis R. van den Brink-van der Laan E. de Kruijff B. Wallace B.A. Palmer T. Berks B.C. Biochemistry. 2002; 41: 13690-13697Crossref PubMed Scopus (96) Google Scholar, 15Oates J. Barrett C.M. Barnett J.P. Byrne K.G. Bolhuis A. Robinson C. J. Mol. Biol. 2005; 346: 295-305Crossref PubMed Scopus (94) Google Scholar, 16Gohlke U. Pullan L. McDevitt C.A. Porcelli I. de Leeuw E. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10482-10486Crossref PubMed Scopus (219) Google Scholar). Low resolution single particle electron microscopy studies of detergent-solubilized TatA complexes show ring-shaped structures with inner diameters suitable for accommodating typical Tat transported substrates (16Gohlke U. Pullan L. McDevitt C.A. Porcelli I. de Leeuw E. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10482-10486Crossref PubMed Scopus (219) Google Scholar, 17Tarry M.J. Schäfer E. Chen S. Buchanan G. Greene N.P. Lea S.M. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13284-13289Crossref PubMed Scopus (79) Google Scholar). A lid structure is associated with one side of the ring pore. The TatB and TatC proteins form a separate large, hetero-oligomeric complex in detergent solution (17Tarry M.J. Schäfer E. Chen S. Buchanan G. Greene N.P. Lea S.M. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13284-13289Crossref PubMed Scopus (79) Google Scholar, 18Bolhuis A. Mathers J.E. Thomas J.D. Barrett C.M. Robinson C. J. Biol. Chem. 2001; 276: 20213-20219Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Current models for the mechanism of Tat translocation suggest that substrate proteins initially bind to the TatBC complex, which then recruits TatA to form the active translocation site (17Tarry M.J. Schäfer E. Chen S. Buchanan G. Greene N.P. Lea S.M. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13284-13289Crossref PubMed Scopus (79) Google Scholar, 19Cline K. Mori H. J. Cell Biol. 2001; 154: 719-729Crossref PubMed Scopus (244) Google Scholar, 20Alami M. Lüke I. Deitermann S. Eisner G. Koch H.G. Brunner J. Müller M. Mol. Cell. 2003; 12: 937-946Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 21Leake M.C. Greene N.P. Godun R.M. Granjon T. Buchanan G. Chen S. Berry R.M. Palmer T. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 15376-15381Crossref PubMed Scopus (148) Google Scholar, 22Dabney-Smith C. Cline K. Mol. Biol. Cell. 2009; 20: 2060-2069Crossref PubMed Scopus (54) Google Scholar, 23Mori H. Cline K. J. Cell Biol. 2002; 157: 205-210Crossref PubMed Scopus (197) Google Scholar, 24Dabney-Smith C. Mori H. Cline K. J. Biol. Chem. 2006; 281: 5476-5483Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). TatA is thought to form the protein-conducting element of the system. The task faced by TatA is challenging, because it must allow the transport of large protein substrates of varying sizes while maintaining the membrane permeability barrier to ions and small molecules. There is only limited information about how this is achieved. There is suggestive evidence that TatA may polymerize in response to substrate (21Leake M.C. Greene N.P. Godun R.M. Granjon T. Buchanan G. Chen S. Berry R.M. Palmer T. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 15376-15381Crossref PubMed Scopus (148) Google Scholar, 22Dabney-Smith C. Cline K. Mol. Biol. Cell. 2009; 20: 2060-2069Crossref PubMed Scopus (54) Google Scholar), and it has been suggested that the oligomeric state of TatA may dynamically alter to accommodate substrate proteins of different sizes (16Gohlke U. Pullan L. McDevitt C.A. Porcelli I. de Leeuw E. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10482-10486Crossref PubMed Scopus (219) Google Scholar). However, to fully understand how TatA complexes are able to mediate the transmembrane movement of folded proteins structural information at the molecular level of resolution is now required. In this study we have used site-directed spin labeling to probe the structure and organization of TatA subunits. Site-directed spin labeling is a powerful method to obtain structural information on proteins, such as TatA, that are not easily studied by crystallographic or solution NMR methods (25Hubbell W.L. Gross A. Langen R. Lietzow M.A. Curr. Opin. Struct. Biol. 1998; 8: 649-656Crossref PubMed Scopus (500) Google Scholar, 26Hubbell W.L. Cafiso D.S. Altenbach C. Nat. Struct. Biol. 2000; 7: 735-739Crossref PubMed Scopus (724) Google Scholar, 27Columbus L. Hubbell W.L. Trends Biochem. Sci. 2002; 27: 288-295Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 28Perozo E. Cortes D.M. Sompornisut P. Kloda K. Martinac B. Nature. 2002; 418: 942-948Crossref PubMed Scopus (494) Google Scholar). The room temperature EPR spectra of a covalently attached spin label is sensitive to the dynamics of the label and hence its protein environment (29Hubbell W.L. Altenbach C. Curr. Opin. Struct. Biol. 1994; 4: 566-573Crossref Scopus (364) Google Scholar, 30Hubbell W.L. Mchaourab H.S. Altenbach C. Lietzow M.A. Structure. 1996; 4: 779-793Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 31Borbat P.P. Costa-Filho A.J. Earle K.A. Moscicki J.K. Freed J.H. Science. 2001; 291: 266-269Crossref PubMed Scopus (291) Google Scholar). Thus a set of spin labels along a helix can provide a powerful means of deducing whether a residue is sitting at the protein surface or at a buried site. Unpaired electron spins will also interact, via dipolar coupling, with a nearby spin. The magnitude of this coupling is proportional to 1/rab3, where rab is the inter-spin distance. Measurements of inter-spin coupling can, therefore, be used to estimate the distances between the spins and hence the distances between different regions of spin-labeled proteins (32Steinhoff H.J. Radzwill N. Thevis W. Lenz V. Brandenburg D. Anston A. Dodson G. Wollmer A. Biophys. J. 1997; 73: 3287-3298Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 33Persson M. Harbridge J.R. Hammarström P. Mitri R. Mårtensson L.G. Carlsson U. Eaton G.R. Eaton S.S. Biophys. J. 2001; 80: 2886-2897Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 34Radzwill N. Gerwert K. Steinhoff H.J. Biophys. J. 2001; 80: 2856-2866Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In this study we have carried out EPR measurements on purified, detergent-solubilized TatA complexes containing site-directed nitroxide spin labels at consecutive positions within the transmembrane helix. The results suggest a model for the organization of the TatA complex in which a single wall of transmembrane helices interact via residues Ile12 and Val14. We also observed subunit exchange between TatA complexes. This is consistent with models for the mechanism of Tat transport that invoke dynamic TatA polymerization. Plasmid pQE80-TatA expresses E. coli TatA with a carboxyl-terminal hexahistidine tag. pQE80-TatA was produced by amplifying the tatA gene from plasmid pUNITATA (35Greene N.P. Porcelli I. Buchanan G. Hicks M.G. Schermann S.M. Palmer T. Berks B.C. J. Biol. Chem. 2007; 282: 23937-23945Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) using the primers TATA5 (11Sargent F. Bogsch E.G. Stanley N.R. Wexler M. Robinson C. Berks B.C. Palmer T. EMBO J. 1998; 17: 3640-3650Crossref PubMed Scopus (443) Google Scholar) and TatA_ RS_His(5′-ATACGTGGATCCTTAGTGATGGTGATGGTCATGAGATCTCACCTGCTCTTTATCGTGG-3′), digesting the product with EcoRI and BamHI, and cloning into the same sites in pQE80 (Qiagen). Plasmids expressing single cysteine TatA variants were constructed by using the same strategy as pQE80-TatA but with the appropriate mutant tatA alleles amplified from the pUNITATA-derived plasmids described by Greene et al. (35Greene N.P. Porcelli I. Buchanan G. Hicks M.G. Schermann S.M. Palmer T. Berks B.C. J. Biol. Chem. 2007; 282: 23937-23945Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). E. coli strain C43 (36Miroux B. Walker J.E. J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1578) Google Scholar) transformed with the appropriate expression plasmid was cultured aerobically at 37 °C in Luria-Bertani (LB) medium. When the cultures reached an A600 nm of 0.5, expression of the tatA allele was routinely induced with 1 mm final concentration of isopropyl β-d-thiogalactoside, and the growth continued for a further 5 h before harvesting. The TatA Ile11 → Cys variant did not express well under these conditions. Instead, production of this variant was induced by 0.5 mm final concentration isopropyl β-d-thiogalactoside, and the cells were cultured for a further 20 h at 25 °C. Cells were harvested by centrifugation and resuspended in 20 mm MOPS, pH 7.2 (at 25 °C), 200 mm NaCl (buffer A) containing 2 mm dithiothreitol. Isolation of a crude membrane fraction, solubilization of the membranes with nonapolyoxyethylene dodecyl ether (C12E9, Sigma), and purification of TatA variants from the membrane extract by Ni(II) affinity chromatography were as described earlier (14Porcelli I. de Leeuw E. Wallis R. van den Brink-van der Laan E. de Kruijff B. Wallace B.A. Palmer T. Berks B.C. Biochemistry. 2002; 41: 13690-13697Crossref PubMed Scopus (96) Google Scholar) with the following modifications. A 5-ml HisTrap Chelating HP column (Amersham Biosciences) was used in the affinity purification step. 2 mm dithiothreitol was included in the buffer at all steps up to and including the Ni(II) affinity column wash step to ensure that the cysteine residue of the TatA variants was in the reduced state. Dithiothreitol was omitted from the affinity column elution step onward to avoid interference with the labeling reaction. TatA-containing fractions from the Ni(II) affinity column were pooled and incubated for 16 h at 4 °C and then 4 h at room temperature with 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl (MTSL, Toronto Research Chemicals, Canada). A 10:1 molar excess of MTSL over TatA was employed to produce fully labeled protein, while substoichiometric MTSL:TatA ratios were used for method 1 underlabeling. The sample was then supplemented with Na2EDTA to a final concentration of 20 mm and concentrated to a volume of 0.5 ml using a 100-kDa molecular mass cut-off centrifugal concentrator (Millipore). The concentrated sample was subjected to size-exclusion chromatography on a Superose 6 10/300 GL column (Amersham Biosciences) in buffer A containing 0.1% C12E9. TatA-containing fractions were identified by SDS-PAGE, pooled, dialyzed against buffer A containing 0.1% C12E9, and concentrated to ∼5 mg of protein ml−1 using a 100-kDa molecular mass cut-off microcon centrifugal concentrator (Millipore). Protein concentrations were estimated using the Bio-Rad protein assay with bovine serum albumin as the standard. The degree of labeling was determined in each case by comparing the measured protein concentration to the spin label concentration obtained by integration of the spin intensity of the EPR spectrum. The purified MTSL-labeled TatA variants were analyzed on a Bruker Ultraflex TOF/TOF matrix-assisted laser desorption ionization time-of-flight mass spectrometer, using a sinapinic acid matrix. All variants showed a major peak of mass within 5 Da of that expected for TatA-MTSL. Analysis of mass peak intensities suggested >90% MTSL labeling for all “fully labeled” TatA variants in agreement with the biochemical analysis. Room temperature X-band EPR spectra were acquired using a Bruker EleXsys 500 spectrometer fitted with an ER4123D resonator with samples contained in 0.6-mm inner diameter × 0.84-mm outer diameter quartz tubes (37Carl P. The ER4123D CW-Resonator: Dedicated to Spin Labels.Bruker BioSpin Application Note. Bruker BioSpin, Coventry, UK2003Google Scholar). A Bruker EleXsys 580 system with an ER4118X-MD resonator was used in the continuous-wave mode to record X-band EPR at 170 K. For this system 3-mm inner diameter × 4-mm outer diameter quartz tubes were used. W-band EPR spectra were acquired using a Bruker, EleXsys 680 spectrometer with samples contained in 0.1-mm inner diameter × 0.5-mm outer diameter quartz tubes. Samples of TatA were prepared with nitroxide radical spin labels at each of positions 9–18. These residues encompass the center and carboxyl-terminal half of the transmembrane helix and correspond to just over 2.5 helical turns. We chose this region of TatA for analysis, because previous studies had shown that this part of TatA forms well defined interactions with other TatA protomers and because cysteine substitutions at the chosen positions do not significantly affect TatA function (35Greene N.P. Porcelli I. Buchanan G. Hicks M.G. Schermann S.M. Palmer T. Berks B.C. J. Biol. Chem. 2007; 282: 23937-23945Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Appropriate hexahistidine-tagged single cysteine TatA variants were purified in the detergent C12E9 and then labeled with the thiol-specific spin label MTSL (38Berliner L.J. Ann. N. Y. Acad. Sci. 1983; 414: 153-161Crossref PubMed Scopus (55) Google Scholar, 39Hubbell W.L. Altenbach C. White S.H. Membrane Protein Structure: Experimental Approaches. Oxford University Press, London, UK1994: 224-248Google Scholar, 40Feix J.B. Klug C.S. Berliner L.J. Spin Labeling: The Next Millennium, Biological Magnetic Resonance. Vol. 14. Plenum Press, New York1998: 251-281Google Scholar). The purification procedure was essentially identical to the one used previously to obtain low resolution structures of TatA by electron microscopy (16Gohlke U. Pullan L. McDevitt C.A. Porcelli I. de Leeuw E. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10482-10486Crossref PubMed Scopus (219) Google Scholar). Thus the structural data from the two studies should be directly comparable. Blue native-PAGE analysis (41Schägger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1910) Google Scholar) of the purified spin-labeled variants revealed ladders of bands resembling those exhibited by the wild-type TatA protein (15Oates J. Barrett C.M. Barnett J.P. Byrne K.G. Bolhuis A. Robinson C. J. Mol. Biol. 2005; 346: 295-305Crossref PubMed Scopus (94) Google Scholar, 16Gohlke U. Pullan L. McDevitt C.A. Porcelli I. de Leeuw E. Palmer T. Saibil H.R. Berks B.C. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10482-10486Crossref PubMed Scopus (219) Google Scholar), and spin-labeled protein was eluted in the same fractions in gel filtration as the wild-type TatA (14Porcelli I. de Leeuw E. Wallis R. van den Brink-van der Laan E. de Kruijff B. Wallace B.A. Palmer T. Berks B.C. Biochemistry. 2002; 41: 13690-13697Crossref PubMed Scopus (96) Google Scholar). To confirm that the spin labels were attached only at the engineered sites, a batch of cysteine-free parental TatA was taken through the same procedure. This resulted in an EPR spectrum devoid of any features from a nitroxide spin label (Fig. 1A). The EPR line shapes recorded at room temperature for the spin-labeled TatA variants contain contributions not only from the motions of the spin label and protein but also from any inter-spin dipolar coupling. Separation of these two effects is necessary to interpret the EPR spectra. Freezing the sample removes the contributions from label and protein motions allowing the analysis of dipolar coupling. Conversely, any contribution from dipolar coupling can be reduced by diluting spins thereby increasing inter-spin distances. In this study two different methods of sub-stoichiometric labeling were used to obtain spin dilution. In method 1 TatA was underlabeled by adding substoichiometric amounts of MTSL to the protein during the labeling reaction. In method 2 fully labeled TatA was mixed with unlabeled wild-type TatA. Identical line shape broadening was seen whichever spin dilution method was employed, and there was no significant difference between 30%, 20, and 10% dilutions. Therefore we assumed that 10% spin dilution is sufficient to remove effects due to dipolar coupling. An example is given in Fig. 1 where the room temperature EPR spectrum of 100% spin-labeled variant Ile-15 → Cys (Fig. 1B) is compared with 10% spin-labeled protein prepared by underlabeling (method 1, Fig. 1C) or prepared by mixing with wild-type protein (method 2, Fig. 1D). Line shape broadening can be characterized by a parameter R, which is the ratio of the height of the low field peak to that of the central resonance line. Both 10% labeled samples have R = 0.45 and, therefore, show identical line shape broadening. The equivalence of the two spin dilution methods is an important observation, because TatA subunits can only have the same environment after mixing that they do after underlabeling if the subunits equilibrate completely between TatA complexes. If such subunit exchange had not occurred then method 2 would be expected to yield a 9:1 mixture of completely unlabeled and 100% labeled complexes resulting in an EPR spectrum identical to that of a stoichiometrically labeled sample but with a 10-fold reduction in spin intensity. Our data show that subunit exchange was complete by the time the samples had been mixed and an EPR spectrum recorded (about 10 min). Thus the rate of helical exchange is relatively rapid. To investigate the kinetics of exchange in more detail samples were flash-frozen in liquid nitrogen at different times after mixing, and spectral broadening was measured in the low temperature EPR spectrum (data not shown). These experiments showed that subunit exchange was effectively complete at the shortest measurable mixing time (1 min). Nitroxide motional dynamics cause spectral broadening in the room temperature EPR of spin-labeled proteins. Various molecular motions contribute to probe mobility, including the flexibility of the spin-labeled side chain relative to the protein, local protein backbone fluctuations, and the overall rotary diffusion of the protein (27Columbus L. Hubbell W.L. Trends Biochem. Sci. 2002; 27: 288-295Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). At 9.5 GHz and room temperature the rotational diffusion of oligomeric complexes of TatA will be too slow on the EPR timescale to affect the line shape and can be ignored. By contrast, internal rotations of the spin label around the bonds in its flexible linker, and backbone motions of the protein itself, can both be sufficiently rapid (τ ∼ 1 ns) to influence spectral line shape (26Hubbell W.L. Cafiso D.S. Altenbach C. Nat. Struct. Biol. 2000; 7: 735-739Crossref PubMed Scopus (724) Google Scholar). These motions and their anisotropy will be strongly influenced by packing interactions in the local environment and by the backbone secondary structure. Spectral line shape analysis can, therefore, show whether the spin label is at a buried or surface site and give information about the type of secondary structure in which the spin label is located. Semi-empirical measures of spin label mobility can be derived from the EPR “mobility” parameter ΔH0, measured as the peak-to-peak first derivative width of the central line, and by the spectral breadth parameter, which is represented by the second moment of the spectral breadth, 〈H2〉 (42Mchaourab H.S. Lietzow M.A. Hideg K. Hubbell W.L. Biochemistry. 1996; 35: 7692-7704Crossref PubMed Scopus (532) Google Scholar, 43Bordignon E. Steinhoff H.-J. Hemminga M.A. Berliner L.J. Membrane Protein Structure and Dynamics. Springer, New York2007: 129-164Google Scholar), 〈H2〉=∫(B-〈H〉)2S(B)dB∫S(B)dB(1) where B is magnetic field, S(B) is the integrated absorbance of the EPR spectrum of the spin label, and the first moment 〈H〉 = ∫B S(B)dB/∫S(B)dB. Using proteins of known structure, correlations between the inverse values of these parameters, ΔH0−1 and 〈H2〉−1, allow distinction between residues that are buried, surface-exposed, in a loop, or at contact sites between secondary structure regions (43Bordignon E. Steinhoff H.-J. Hemminga M.A. Berliner L.J. Membrane Protein Structure and Dynamics. Springer, New York2007: 129-164Google Scholar). Room temperature X-band EPR spectra were collected for the TatA variants successively spin-labeled from residue Leu-9 to Leu-18 (Fig. 2), and spin mobility parameters were calculated (Fig. 3). In each case the protein was 10% spin-labeled by dilution of 100% labeled TatA with wild-type TatA. As discussed in the previous section this removes any detectable broadening from interspin dipolar coupling and hence the EPR line shapes reflect only the motional dynamics of each spin label. Fig. 3A shows the variation of side-chain mobility, represented by ΔH0−1 values, along the transmembrane helix. Positions 12 and 14 have the most restricted mobility, whereas positions 9, 10, and 18 are significantly more mobile than the average value of 2.8 millitesla−1. This suggests two sites of significant inter-protein contact at the center of the region probed, namely residues 12 and 14. By plotting 〈H2〉−1 against ΔH0−1 for each sequence position residues can be assigned to regions of secondary structure and of inter-protein contact (Fig. 3B). This plot suggests that residues 12 and 14 are at contact interfaces, whereas residues 11, 13, and 15–17 are exposed at a helical surface.FIGURE 3Mobility of spin labels attached to the transmembrane helix of TatA. A, variation of the mobility parameter ΔH0−1 along the transmembrane helix of TatA. The ΔH0−1 values were calculated from the room temperature EPR spectra given in Fig. 2. Error bars are calculated from the results of duplicate sample preparations. B, correlation of the mobility parameters 〈H2〉−1 (inverse second moment) and ΔH0−1 (inverse of the central line width) for spin labels at positions 9–18 within TatA. Mobility parameters were calculated from the room temperature X-band EPR spectra shown in Fig. 2. Assignment of structural environments is based on the compilation of Bordignon and Steinhoff (43Bordignon E. Steinhoff H.-J. Hemminga M.A. Berliner L.J. Membrane Protein Structure and Dynamics. Springer, New York2007: 129-164Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In frozen solution the motions of the spin labels are quenched, and any broadening in EPR line shape is due to dipolar coupling between labels. The strength of the dipolar coupling depends on the separation between the interacting labels and thus gives information on the distance between the labeled positions. If the interacting spin separation is ∼5 Å or less the dipolar coupling may become large in comparison with the energy of the microwave photon, rendering the interacting spins EPR silent. X-band EPR spectra were collected for the ten spin-labeled TatA variants at 170 K for both 10 and 100% labeled samples. Fig. 4A shows the spectra for a representative variant. The changes in line shape on dilution are characteristic of those observed in the presence of weak magnetic dipolar interactions between nitroxide spin labels (32Steinhoff H.J. Radzwill N. Thevis W. Lenz V. Brandenburg D. Anston A. Dodson G. Wollmer A. Biophys. J. 1997; 73: 3287-3298Abstract Full Text PDF PubMed Scopus (149) Google Scholar). The broadening in the 100% labeled spectrum is similar for all label positions between 9 and 18. Thus the distance between the same positions in adjacent protomers is approximately the same for all positions analyzed. Crucially, comparison of the integrated EPR signal intensities of the fully labeled and underlabeled samples shows no loss of spin concentration with increased labeling. Thus there is no evidence for loss of signal intensity that wo" @default.
• W2079392253 created "2016-06-24" @default.
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• W2079392253 creator A5048730287 @default.
• W2079392253 creator A5050528640 @default.
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• W2079392253 date "2010-01-01" @default.
• W2079392253 modified "2023-09-27" @default.
• W2079392253 title "Subunit Organization in the TatA Complex of the Twin Arginine Protein Translocase" @default.
• W2079392253 cites W1604304986 @default.
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