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W2077305297 abstract "Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) fromMethylophilus methylotrophus (sp. W3A1). Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution. These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible “hinge” sequences that are unique to M. methylotrophus ETF. The x-ray scattering data are consistent with previous models concerning the interaction of M. methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase. Our data reveal that an “induced fit” mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process. Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) fromMethylophilus methylotrophus (sp. W3A1). Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution. These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible “hinge” sequences that are unique to M. methylotrophus ETF. The x-ray scattering data are consistent with previous models concerning the interaction of M. methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase. Our data reveal that an “induced fit” mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process. electron-transferring flavoprotein flavin-adenine dinucleotide trimethylamine dehydrogenase electron transfer small angle x-ray scattering polymerase chain reaction Electron-transferring flavoproteins (ETFs)1 act as physiological electron carriers between degradative enzymes in bacteria and mitochondria and their respective membrane-bound electron transport pathways (1.Thorpe C. Muller F. Chemistry and Biochemistry of Flavoenzymes. II. CRC Press, Boca Raton, FL1991: 471-486Google Scholar). ETFs are heterodimeric, and they all possess one equivalent of non-covalently bound flavin-adenine dinucleotide (FAD) per ETF dimer, with the exception of ETF from Megasphaera elsdenii, which contains 2 equivalents of FAD per dimer (2.Whitfield C.D. Mayhew S.G. J. Biol. Chem. 1974; 249: 2801-2810Abstract Full Text PDF PubMed Google Scholar). The ETFs from pig, human, Paracoccus denitrificans, andMethylophilus methylotrophus also contain 1 equivalent of AMP per protein dimer (3.Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (153) Google Scholar, 4.Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.-J.P. Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (56) Google Scholar, 5.Sato K. Nishina Y. Shiga K. J. Biochem. 1993; 114: 215-222Crossref PubMed Scopus (40) Google Scholar, 6.Duplessis E.R. Rohlfs R.J. Hille R. Thorpe C. Biochem. Mol. Biol. Int. 1994; 32: 195-199PubMed Google Scholar). ETF from M. methylotrophus(sp. W3A1) is a highly specific electron carrier that accepts electrons from only one enzyme, trimethylamine dehydrogenase (TMADH) (7.Steenkamp D.J. Gallup M. J. Biol. Chem. 1978; 253: 4086-4089Abstract Full Text PDF PubMed Google Scholar). In methylotrophic bacteria, TMADH catalyzes the oxidative demethylation of trimethylamine to dimethylamine and formaldehyde (8.Steenkamp D.J. Mallinson J. Biochim. Biophys. Acta. 1976; 429: 705-719Crossref PubMed Scopus (56) Google Scholar). The enzyme enables these organisms to subsist on trimethylamine as their sole source of carbon. M. methylotrophus ETF consists of two subunits with molecular masses of 34 and 29 kDa (9.Chen D.W. Swenson R.P. J. Biol. Chem. 1994; 269: 32120-32130Abstract Full Text PDF PubMed Google Scholar), and it shares considerable sequence identity with bacterial and mammalian ETFs (3.Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (153) Google Scholar, 4.Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.-J.P. Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (56) Google Scholar). Preliminary crystallographic studies of M. methylotrophus ETF have been reported, but to date no crystallographic structure for the protein is available (10.White S.A. Mathews F.S. Rohlfs R.J. Hille R. J. Mol. Biol. 1994; 240: 265-266Crossref PubMed Scopus (10) Google Scholar). Mammalian and bacterial ETFs are thought to act as 1-electron carriers, cycling between the oxidized and anionic flavin semiquinone forms. The ETF from M. elsdenii is unusual in that it serves as a 2-electron carrier. ETFs from mammalian sources and P. denitrificans can be reduced (albeit over a long time period) to the dihydroquinone form by reduction with dithionite or during photochemical reduction. M. methylotrophus ETF is readily converted to the semiquinone form in reactions with TMADH or during artificial reduction with dithionite. Further reduction of M. methylotrophus ETF to its dihydroquinone state can be accomplished electrochemically (11.Byron C.M. Stankovich M.T. Husain M. Davidson V.L. Biochemistry. 1989; 28: 8582-8587Crossref PubMed Scopus (36) Google Scholar) or when ETF is in complex with TMADH (12.Jang M.-H. Scrutton N.S. Hille R. J. Biol. Chem. 2000; 275: 12546-12552Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). That ETF can be reduced to the 2-electron level when in complex with TMADH provides evidence for structural perturbation on forming the electron transfer complex. The midpoint reduction potentials of the quinone/semiquinone and semiquinone/dihydroquinone couples of the FAD in M. methylotrophus ETF have been determined (11.Byron C.M. Stankovich M.T. Husain M. Davidson V.L. Biochemistry. 1989; 28: 8582-8587Crossref PubMed Scopus (36) Google Scholar). The potential of the quinone/semiquinone couple is exceptionally high (+196 mV), consistent with a need to accept electrons from the 4Fe-4S center of TMADH (midpoint potential, +102 mV (13.Barber M.J. Pollock V. Spence J.T. Biochem. J. 1988; 256: 657-659Crossref PubMed Scopus (24) Google Scholar)). The semiquinone/dihydroquinone couple, however, is more conventional in value (−197 mV), indicating that there is a kinetic block on full reduction of ETF by dithionite (−530 mV) or photoexcited deazariboflavin (−650 mV). The potentials measured for free ETF may not of course reflect the situation in the electron transfer complex but nevertheless are likely to serve as a reasonable guide. The structure of human ETF has been determined at 2.1-Å resolution (3.Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (153) Google Scholar), and this protein shares 31% sequence identity with M. methylotrophus ETF. Using the x-ray structure of human ETF as a template, a model of the structure of M. methylotrophus ETF has been built both in free solution and in complex with TMADH (14.Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). The model predicts that the two subunits (subunit α (residues 1–321) and subunit β (residues 322–585)) of M. methylotrophusETF comprise three domains, viz. domain I (the N-terminal region of the α subunit), domain II (the C terminus of the α subunit and a small C-terminal region of the β subunit), and domain III (the majority of the β subunit). M. methylotrophus ETF is thought to be Y-shaped, with domains I and III forming a shallow “bowl” in which domain II rests; domain II is thought to be connected to domains I and III by two flexible regions of polypeptide chain (14.Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). The isoalloxazine ring of FAD interacts almost exclusively with domain II (see Fig. 1). Significantly, the model shows that domain II of the M. methylotrophus ETF model must be rotated by approximately 50° relative to domains I and III in order to form an active electron transfer complex with TMADH (14.Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar) (Fig.1). The models of ETF and the electron transfer complex formed between TMADH and ETF suggest that ETF is a dynamic molecule and that large scale conformational changes are required for complex assembly. It has been proposed that free ETF adopts a discrete electron transfer (eT)-inactive conformation similar to that of crystalline human ETF, whereas in complex with TMADH, ETF adopts an eT-active conformation. The possibility arises, however, that free ETF can populate a range of conformations between the eT-inactive and eT-active forms and that these are converted to the eT-active conformation during complex assembly. In this paper we have used small angle x-ray scattering (SAXS) experiments to gain additional insight into the structural properties of M. methylotrophus ETF in solution. This low resolution technique is a powerful method to probe the arrangement of domains in multidomain proteins, protein oligomerization, and complex formation (see e.g. Ref. 15.Feigin L.A. Svergun D.I. Structure Analysis by Small-Angle X-Ray and Neutron Scattering. Plenum Publishing Corp., New York1987Crossref Google Scholar). Moreover, the use of SAXS experiments to obtain ab initio model-independent molecular envelope structures of proteins in solution has been described (16.Svergun D.I. Volkov V.V. Kozin M.B. Stuhrmann H.B. Barberato C. Koch M.H.J. J. Appl. Crystallogr. 1997; 30: 798-802Crossref Google Scholar, 17.Grossmann J.G. Hasnain S.S. J. Appl. Crystallogr. 1997; 30: 770-775Crossref Google Scholar) as an effective approach for identifying molecular features and changes in soluble proteins. The method, for example, has been used successfully to explore the large conformational change in metal-bound and metal free transferrin (18.Grossmann J.G. Crawley J.B. Strange R.W. Patel K.J. Murphy L.M. Neu M. Evans R.W. Hasnain S.S. J. Mol. Biol. 1998; 279: 461-472Crossref PubMed Scopus (53) Google Scholar) and the methane monooxygenase electron transfer complex (19.Gallagher S.C. Callaghan A.J. Zhao J. Dalton H. Trewhella J. Biochemistry. 1999; 38: 6752-6760Crossref PubMed Scopus (36) Google Scholar). Here we demonstrate that our SAXS data are consistent with domain II of M. methylotrophus ETF pivoting around the predicted hinge regions at the domain II/domain I and domain II/domain III boundaries and that complex assembly with TMADH must therefore proceed by an induced fit mechanism. The region of M. methylotrophus genomic DNA encoding the two subunits of ETF was amplified using the polymerase chain reaction (PCR). Oligonucleotide primers 1 (5′ AAT GAA GGA GAC GAA GGT ATG AAG ATA TTA GTG 3′) and 2 (5′ TTT TTT TTT AAA CTA TGC TGC AAG CTG CGC TTT CAG CTC TTC 3′) were designed using the published gene sequence forM. methylotrophus ETF (9.Chen D.W. Swenson R.P. J. Biol. Chem. 1994; 269: 32120-32130Abstract Full Text PDF PubMed Google Scholar). The PCR was carried out using Vent DNA polymerase as specified by the manufacturer (New England Biolabs, Inc.). PCR products were purified using the Wizard PCR Preps DNA purification system as specified by the manufacturer (Promega) and then phosphorylated at the 5′ termini using T4 polynucleotide kinase (New England Biolabs, Inc.). T4 polynucleotide kinase was removed by phenol/chloroform extraction, and the DNA was concentrated by ethanol precipitation. PCR products were ligated to plasmid pKK223–3 (Amersham Pharmacia Biotech) that had been digested with the restriction enzymeSmaI and treated with calf intestinal alkaline phosphatase to remove the 5′ phosphate group. DNA from the ligation reaction was used to transform the E. coli strain TG1. The correct construct (pKKETF11) was verified by restriction analysis, and the genes encoding ETF were resequenced to ensure that spurious changes had not arisen during the amplification procedure. E. colistrain TG1 transformed with pKKETF11 and grown at 22 °C in 2 × TY medium (10 g of bactotryptone, 10 g of yeast extract, 5 g of NaCl per 1 liter) produced ∼2.5 mg ETF per liter of late exponential phase culture. Improved expression was achieved by deleting the M. methylotrophus ribosome-binding site located before the ETF genes and replacing it with the ribosome-binding site found in the pKK223–3 expression vector. The deletion was performed using the QuikChange site-directed mutagenesis kit supplied by Stratagene and oligonucleotides 3 (5′ CAC ACA GGA AAC AGA ATT CAT GAA GAT ATT AGT GGC AG 3′) and 4 (5′ CTG CCA CTA ATA TCT TCA TGA ATT CTG TTT CCT GTG TG 3′). To ensure that no spurious changes had arisen as a result of the mutagenesis reaction, the entire ETF gene was resequenced using the Amersham Pharmacia Biotech T7 sequencing kit and protocols. Recombinant ETF was expressed from the new plasmid (pED1) in the E. colistrain TG1 at 20 °C on 2 × TY medium supplemented with 50 μg/ml timentin. The protein was purified in large quantities (∼30 mg/liter of late exponential phase culture) as described by Chen and Swenson (9.Chen D.W. Swenson R.P. J. Biol. Chem. 1994; 269: 32120-32130Abstract Full Text PDF PubMed Google Scholar). ETF is purified in the reduced (anionic semiquinone) form. Oxidized ETF for x-ray scattering studies was obtained by treatment with potassium ferricyanide followed by rapid gel filtration (Sephadex 25) to remove excess oxidant. Oxidized protein was used immediately in scattering experiments after gel filtration and dilution (for concentration-dependent scattering experiments). UV-visible absorption spectra were recorded before and after x-ray exposure for samples in both oxidation states to ensure that no x-ray-dependent redox change occurred. This also allowed the concentration of ETF samples to be determined using the absorption at 438 nm (ε438 = 11,300m−1 cm−1) (7.Steenkamp D.J. Gallup M. J. Biol. Chem. 1978; 253: 4086-4089Abstract Full Text PDF PubMed Google Scholar). SAXS experiments were performed with protein concentrations between 0.3 and 15 mg/ml. X-ray solution scattering data were collected in two sessions with the low angle scattering camera on station 2.1 (20.Towns-Andrews E. Berry A. Bordas J. Mant P.K. Murray K. Roberts K. Sumner I. Morgan J.S. Lewis R. Gabriel A. Rev. Sci. Instrum. 1989; 60: 2346-2349Crossref Scopus (137) Google Scholar) at the Synchotron Radiation Source, Daresbury, UK using a position-sensitive multiwire proportional counter (21.Lewis R.R. J. Synchrotron Radiat. 1994; 1: 43-53Crossref PubMed Google Scholar). At the sample-to-detector distance of 2.4 m and the x-ray wavelength of λ = 1.54 Å, a momentum transfer interval of 0.002 Å−1 ≤ s ≤ 0.050 Å−1 was covered. The modulus of the momentum transfer is defined as s = 2 sin Θ/λ, where 2Θ is the scattering angle. The s range was calibrated using an oriented specimen of wet rat tail collagen (based on a diffraction spacing of 670 Å). Samples were contained in a brass cell holding a teflon ring sandwiched by two mica windows that defines a sample volume of 120 μl and a thickness of 1.5 mm. The brass cell was maintained at 4 °C during data acquisition. Buffer and sample were measured in alteration, each in a frame of 60 s (amounting to a total measuring time of up to 30 min depending on sample concentration and changes monitored on-line during experiments). Reduction and analysis of scattering data were performed as described previously (18.Grossmann J.G. Crawley J.B. Strange R.W. Patel K.J. Murphy L.M. Neu M. Evans R.W. Hasnain S.S. J. Mol. Biol. 1998; 279: 461-472Crossref PubMed Scopus (53) Google Scholar). The radius of gyration (Rg), the forward scattering intensity (Io), and the intraparticle distance distribution function p(r) were calculated from the experimental scattering data using the indirect Fourier transform method as implemented in the program GNOM (22.Semenyuk A.V. Svergun D.I. J. Appl. Crystallogr. 1991; 24: 537-540Crossref Scopus (568) Google Scholar). Relative Io /c values (c = sample concentration) give the relative molecular weight of the protein samples when referenced against a suitable standard (bovine serum albumin was used with a known molecular mass of 66 kDa). The maximum linear dimension (D max) of the particle can be evaluated because of the characteristic ofp(r). The volume (V) of the particle can be calculated from the Porod invariant (23.Porod G. Kolloid Z. 1951; 124: 83-114Crossref Scopus (1537) Google Scholar) and a correction factor taking into account the limited experimental scattering range (15.Feigin L.A. Svergun D.I. Structure Analysis by Small-Angle X-Ray and Neutron Scattering. Plenum Publishing Corp., New York1987Crossref Google Scholar). The multipole expansion method proposed by Stuhrmann (24.Stuhrmann H.B. Acta Crystallogr. Sect. A. 1970; 26: 297-306Crossref Scopus (149) Google Scholar) and developed by Svergun et al. (16.Svergun D.I. Volkov V.V. Kozin M.B. Stuhrmann H.B. Barberato C. Koch M.H.J. J. Appl. Crystallogr. 1997; 30: 798-802Crossref Google Scholar) was used to obtain the molecular shape of ETF. The smoothed scattering profile of reduced ETF was fittedab initio by the scattering from an envelope function starting from an ellipsoidal initial approximation (consistent with the experimental Rg and D maxvalues). The molecular shape was characterized with spherical harmonics using 19 free parameters (4th order harmonics), which is acceptable given the minor differences compared with the shape obtained for 3rd order harmonics (10 free parameters) and considering the information content in the data used. To take advantage of the already existing structural knowledge for this three-domain protein, scattering data simulations were carried out using atomic models of the structure of M. methylotrophusETF (produced by homology modeling based on the crystal structure coordinates of human ETF (3.Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (153) Google Scholar)) in free solution and in the conformation expected when in complex with TMADH (14.Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). Parameters and scattering curves were computed from the model coordinates using the program CRYSOL (25.Svergun D. Barberato C. Koch M.H.J. J. Appl. Crystallogr. 1995; 28: 768-773Crossref Scopus (2743) Google Scholar), which also considers the hydration shell of the solvated protein. The solution scattering curves and intraparticle distance distributions for the oxidized and semiquinone forms of M. methylotrophus ETF are shown in Fig.2 A. The figure clearly demonstrates the very close similarity between the scattering profiles for the oxidized and semiquinone forms of the protein. The concentration-dependent low angle scattering region (s ≤ 0.01 Å−1) and radii of gyration are emphasized in Fig. 2 B. The intensity of the scattering profiles at low scattering angles was found to increase as a function of [ETF] for both the oxidized and semiquinone forms of the protein, revealing interparticle interactions. Hence low concentration (≤ 2 mg/ml) measurements were crucial. CarefulI0 analysis established that both forms of ETF behaved as a heterodimeric protein (∼60 kDa) in solution, and aggregation effects and/or radiation damage may have been induced during x-ray exposure (after examination of data collected in time frames). The Rg values obtained by extrapolation to zero concentration for oxidized and semiquinone ETF are virtually identical (26.6 ± 0.4 Å and 26.5 ± 0.4 Å, respectively). Furthermore, the maximum linear dimension and volume of both ETF states revealed equivalent values within error limits (x = 80 Å ± 4%, V = 110,000 Å3 ± 5%). This illustrated that no large conformational transformation had occurred as a result of changing the redox state of ETF. Moreover, thep(r) function with a characteristic shoulder at 40 Å indicates a spread-out, Y-shaped conformation (with distinct domain features) rather than a compact, globular structure. The structure of M. methylotrophus ETF has previously been modeled in two conformations (14.Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). The first conformation (eT-inactive) was obtained from modeling studies in which the structure of M. methylotrophus ETF was built by homology using the crystal coordinates of human ETF (Fig.3 A). The second conformation (eT-active) is that obtained by rotating domain II by 50° with respect to domains I and III such that it produces a complementary fit with the ETF-binding site (26.Wilson E.K. Huang L. Sutcliffe M.J. Mathews F.S. Hille R. Scrutton N.S. Biochemistry. 1997; 36: 41-48Crossref PubMed Scopus (38) Google Scholar) seen in the crystal structure of TMADH (27.Lim L.W. Shamala N. Mathews F.S. Steenkamp D.J. Hamlin R. Xuong N.H. J. Biol. Chem. 1986; 261: 15140-15146Abstract Full Text PDF PubMed Google Scholar) (Fig. 3 B). Using the molecular coordinates for these models, simulated x-ray scattering profiles were generated for each solvated ETF conformer and fitted against the experimental scattering data for M. methylotrophus ETF (Fig. 3 C). The experimental scattering profile for M. methylotrophus ETF does not match agreeably with the simulated profile generated from the structural model for the eT-inactive conformation, which was based on the crystal structure of human ETF. Significant differences in the low angle (s ≤ 0.01 Å−1) and intermediate angle (0.017 Å−1 ≤s ≤ 0.025 Å−1) range result only in a fit with χeT-inactive2 = 2.2 (Fig. 3 C). In contrast, a good fit (χeT-active2 = 1.5) is obtained for the eT-active conformation of M. methylotrophus ETF, which is also reflected in the agreement of calculated and experimental radii of gyration. A good fit (χnew eT-inactive2 = 1.6) is also obtained for the simulated scattering profile of a model of M. methylotrophus ETF in which domain II is rotated 50° toward domain III. The smoothed experimental scattering profile for reducedM. methylotrophus ETF was used to calculate an ab initio low resolution molecular envelope structure for the protein. Molecular shape calculations were carried out up to 4th order harmonics, assuming that the molecule incorporates no axes of symmetry. In all calculations performed, the protein envelope consists of three apparent “domains,” with two of these domains forming a lobed, globular structure on top of which the third domain, a flattened ellipsoid, sits. The theoretical scattering calculated from the restored envelope is given in Fig. 2 as a dotted line (the fit resulted in a residual value of 1.9%). Fig. 4represents the average molecular envelope taking into account several calculations starting from different initial shapes. Manual fitting of the molecular models of M. methylotrophus ETF into this molecular envelope structure resulted in an excellent fit between domains I and III of the model with the two globular domains forming the base of the molecular envelope. With domains I and III of the model fitted to the envelope structure in this orientation, domain II of the model sits at the center of the third domain of the envelope structure, with the latter forming a flattened “halo” around the domain. Previous studies have established that there are spectral changes associated with the binding of oxidized ETF to TMADH (12.Jang M.-H. Scrutton N.S. Hille R. J. Biol. Chem. 2000; 275: 12546-12552Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). These spectral changes, coupled with the ability to reduce ETF to the 2-electron level when in complex with TMADH, were taken as evidence for a structural reorganization of ETF (i.e. induced fit) during complex assembly. The SAXS profiles for both the oxidized and semiquinone forms are very similar, indicating that the solution structure of ETF in both redox states is similar. Difference spectroscopy studies of ETF·TMADH complex assembly with semiquinone ETF also revealed major spectral perturbation (Fig. 5), consistent with both redox forms of ETF undergoing conformational change during complex assembly. It was initially thought that the oxidation state of M. methylotrophus ETF may play a role in the predisposition of the protein to be in either the eT-active or the eT-inactive conformation and that the redox state of the protein would act as a switch between the different conformational states. However, the scattering data indicate that this is not the case, because the SAXS profiles for both oxidized and semiquinone ETF are practically indistinguishable, indicating essentially identical molecular conformations in solution. The fitted experimental SAXS profile for M. methylotrophusETF closely resembled the simulated profile for the eT-active rather than the eT-inactive conformation of ETF, initially leading us to believe that the conformation of M. methylotrophus ETF in solution resembles that of the postulated eT-active form. With regard to protein function, however, it would be unexpected and certainly less beneficial if the protein existed predominantly in its active conformation regardless of its redox state. It would be less beneficial for ETF to exist predominantly in its active confirmation because in the eT-active conformation the isoalloxazine ring is highly exposed, rendering it susceptible to oxidative attack (by molecular oxygen) in the semiquinone redox state. Intriguingly, as a result of the pseudo-symmetry of domains I and III in the M. methylotrophus ETF model, domain II can be rotated by 50° in the direction of domain III. This is contrary to the direction of rotation in converting from the eT-inactive to eT-active conformations (where the rotation is by 50° in the direction of domain I). In terms of the low resolution molecular shape it would be difficult to distinguish between the form of ETF in which the rotation of domain II is toward domain III and the predicted eT-active form (i.e. in which the rotation is toward domain I). Indeed the simulated scattering profile of a model of M. methylotrophus ETF (in which domain II is rotated 50° toward domain III) is as good as the simulated profile for the model of eT-active ETF (Fig. 3 C). The fit to the experimental data results in a χ2 value of 1.6. This additional model for eT-inactive M. methylotrophus ETF (i.e. rotation toward domain III) represents a more “closed” conformation, with the FAD embedded in the interface between domains II and III. However, it is difficult to consider that the protein acts like a conformational switch by going from this more closed, inactive conformation to the eT-active conformation upon oxidation (even though our SAXS results alone cannot completely exclude this possibility). We suggest, not least in view of our spectroscopic studies, that M. methylotrophus ETF may continuously sample this more closed eT-inactive conformation and the eT-active conformation and in doing so also populates other conformations between these two extremes. Such flexibility may account for the difficulties associated with crystallizing M. methylotrophus ETF. Therefore, the uniformity of the observed SAXS profiles (bearing in mind that the scattering measurements represent a time average) for oxidized and semiquinone M. methylotrophus ETF would indicate that this motion is independent of the redox state of ETF. The continuous sampling of a range of conformations between the extremes discussed above is in harmony with the molecular envelope ofM. methylotrophus ETF restored from the experimental SAXS profile. Had there been a sizeable energy barrier between the two extreme conformations (leading to a “flipping” from the eT-inactive to an eT-active conformation and vice versa) one would expect to observe a more triangular molecular envelope structure. This arises because domain II of the model would have to be located adjacent to one or the other of the domains (domains I and III) that form the globular base of the molecule. However, the calculated molecular envelope forms a flattened halo around the locus of domain II of the model. The fit between the molecular envelope structure and domains I and III of the ETF model is clear-cut. Also, domain II of ETF cannot occupy all areas of its corresponding halo simultaneously. Combined, these factors concerning the molecular shape can be taken as evidence to indicate that M. methylotrophus ETF is sampling a range of conformations in solution. In so doing, domain II rotates freely about the axis formed by the polypeptide hinges connecting it to domains I and III. The molecular envelope structure thus represents an average conformation for M. methylotrophus ETF in solution, describing the range of positions occupied by domain II relative to domains I and III. Furthermore, because the halo surrounding domain II of the M. methylotrophus ETF model is flattened in the direction of rotation, it indicates that movement of domain II with respect to domains I and III occurs mainly in one dimension, with very little “side to side” movement. The experimental SAXS data can now be used to refine the original model of domain motion in M. methylotrophus ETF proposed on the basis of homology with the human ETF structure (14.Chohan K.K. Scrutton N.S. Sutcliffe M.J. Protein Pept. Lett. 1998; 5: 231-236Google Scholar). Rather than activation of the ETF·TMADH electron transfer complex being associated with a simple 50° rotation of one of the domains of ETF (i.e. a discrete transition from an eT-inactive to an eT-active conformation) as originally proposed, complex formation stabilizes the eT-active conformation of ETF. During complex formation, the highly dynamic nature of ETF is transiently “frozen” in forming the eT-active conformation. This suppression of the ETF dynamics is achieved by an induced fit mechanism for the interaction of ETF with TMADH. Finally, it is also worth commenting on the comparison between structures of ETF in solution and solid state. Although no crystal structure of M. methylotrophus ETF has been reported yet, atomic structures of the related human and P. denitrificansprotein (3.Roberts D.L. Frerman F.E. Kim J.-J.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14355-14360Crossref PubMed Scopus (153) Google Scholar, 4.Roberts D.L. Salazar D. Fulmer J.P. Frerman F.E. Kim J.-J.P. Biochemistry. 1999; 38: 1977-1989Crossref PubMed Scopus (56) Google Scholar) can be considered as close structural templates. It is therefore interesting to note that the distinct conformation of domain II with respect to domains I and III in the crystalline state (which has been denoted as the eT-inactive state) is in clear contrast to our findings in solution. A close inspection of the molecular arrangements in the crystalline lattice of the two known ETF structures reveals in both cases several close contacts of domain II with domains from neighboring molecules. It is the low energy non-covalent interactions that can effectively influence conformations, particularly in the case of multidomain proteins. Consequently, the crystal packing forces arising from non-covalent bonds are likely to alter or stabilize certain domain orientations. Considering human ETF, structural flexibility would indeed be an asset because the protein has to recognize a whole range of different redox partners suggestive of different protein-protein interactions and docking conformations. By having a dynamic interface this recognition might be achieved more readily. The present study is a good illustration of how crystal structures can be compared with structures investigated under close physiological conditions, i.e. in solution (such as using the solution x-ray scattering technique)." @default.
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