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1、1 Biomolecular solid-state NMR spectroscopy at highest field:the gain in resolution at 1200 MHz Morgane Callon#,1,Alexander A.Malr#,1,Sara Pfister#,1,Vclav Rmal#,1,Marco E.Weber#,1,Thomas Wiegand#,1,Johannes Zehnder#,1,Matas Chvez1,Rajdeep Deb1,Riccardo Cadalbert1,Alexander Dpp1,Marie-Laure Fogeron2
2、,Andreas Hunkeler1,Lauriane Lecoq2,Anahit Torosyan1,Dawid Zyla3,Rudolf Glockshuber3,Stefanie Jonas3,Michael Nassal4,Matthias Ernst*,1,Anja Bckmann*,2,Beat H.Meier*,1 1Physical Chemistry,ETH Zurich,8093 Zurich,Switzerland 2Molecular Microbiology and Structural Biochemistry,UMR 5086 CNRS/Universit de
3、Lyon,69367 Lyon,France 3Institute of Molecular Biology and Biophysics,ETH Zurich,8093 Zurich,Switzerland 4Dept.of Medicine II/Molecular Biology,University of Freiburg 2 Abstract Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths
4、 leading to improved resolution and sensitivity of the NMR spectra.Recently,persistent superconducting magnets at a magnetic field strength(magnetic induction)of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available.We present here a collection of high-field NMR s
5、pectra of a variety of proteins,including molecular machines,membrane proteins and viral capsids and others.We show this large panel in order to provide an overview over a range of representative systems under study,rather than a single best performing model system.We discuss both carbon-13 and prot
6、on-detected experiments,and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.CC-BY-NC-ND 4.0 International licensemade available under a(w
7、hich was not certified by peer review)is the author/funder,who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprintthis version posted March 31,2021.;https:/doi.org/10.1101/2021.03.31.437892doi:bioRxiv preprint 3 Introduction New technologi
8、es have often stood at the beginning of new spectroscopic techniques and NMR is a particularly good example:Microcomputers have enabled Fourier spectroscopy(Ernst and Anderson 1965)and multidimensional NMR(Aue,Bartholdi,and Ernst 1976),high and stable magnetic fields generated by persistent supercon
9、ducting magnets have been instrumental for the first protein structure determinations(Wthrich 2003;Williamson,Havel,and Wuthrich 1985)and the structural and dynamic investigation of increasingly larger proteins(Rosenzweig and Kay 2014;Pervushin et al.1997;Fiaux et al.2002).Reliable magic-angle sampl
10、e spinning probes together with high magnetic fields have enabled biomolecular solid-state NMR spectroscopy(McDermott et al.2000).The first solid-state NMR protein-structure determination used a magnetic-field strength of 17.6 T(proton resonance frequency 750 MHz)(Castellani et al.2002),and the firs
11、t prion fibril structure was determined at 850 MHz(Wasmer et al.2008).A next achievement with important impact was the development of fast magic-angle spinning(MAS)probes,in excess of 100 kHz rotation frequency,enabling proton detection and a reduction of the required sample amount by roughly two or
12、ders of magnitude(Agarwal et al.2014;Andreas et al.2015;Barbet-Massin et al.2014;Schledorn et al.2020;Penzel et al.2019;Lecoq et al.2019).Since 1000 MHz proton Larmor frequency is the present limit of what could be achieved with low-temperature superconducting(LTS)wire(such as Nb3Sn and NbTi),persis
13、tent magnetic fields exceeding 1000 MHz required solenoid coils made out of high-temperature superconducting(HTS)wire(e.g.REBCO)(Maeda and Yanagisawa 2019).Thus,after the highest LTS magnet(1 GHz),it has taken more than five years to develop this new technology and achieve higher fields.Today,persis
14、tent hybrid superconducting magnets combining both,LTS and HTS,have been developed by Bruker Switzerland AG generating magnetic-field strengths up to 28.2 T corresponding to 1200 MHz proton Larmor frequency.What improvement in resolution and sensitivity do we expect by an increase in magnetic field
15、from 850 to 1200 MHz?Assuming that the NMR linewidths are dominated by scalar couplings or residual dipolar couplings under MAS,they should be field-independent when expressed in frequency units(Hz).Then,resolution in NMR spectra benefits when going from 850 to 1200 MHz through an increase in chemic
16、al-shift dispersion(in Hz)by a factor of nearly 1.5(the ratio of the two magnetic fields).On the ppm scale,the linewidth decreases linearly with increasing B0 by the same factor of around 1.5(see Figure S1 for an illustration).With respect to.CC-BY-NC-ND 4.0 International licensemade available under
17、 a(which was not certified by peer review)is the author/funder,who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprintthis version posted March 31,2021.;https:/doi.org/10.1101/2021.03.31.437892doi:bioRxiv preprint 4 sensitivity,the theoret
18、ical gain in signal-to-noise ratio(SNR)is given by!,#$!,%&!/$(Abragam 1961),which corresponds to a factor of 1.7 in the integral of the peaks.These considerations apply both to 13C-and 1H-detected experiments.The above values are valid for“perfect”samples,which do neither show conformational disorde
19、r(resulting in heterogenous line broadening),nor dynamics(resulting in homogenous line broadening).Heterogeneous line broadening scales up linearly with the magnetic field.This contribution to the total linewidth is independent of B0,and stays constant in ppm.In real samples,both disorder and dynami
20、cs can represent important contributions to the linewidths;this is why it is important to illustrate the gain achieved for a broad selection of samples.Besides these sample-dependent effects,several instrumental imperfections can limit the quality of the spectra,including magnetic-field inhomogeneit
21、y in space(shims)and in time(field drifts),or imperfect or unstable magic-angle adjustment and radiofrequency field(rf)inhomogeneity.There are a number of intrinsic challenges when going to higher fields:the larger chemical-shift dispersion makes the application of higher power pulses necessary to c
22、over the entire spectrum;at the same time,obtaining high radio-frequency(rf)fields becomes more demanding at higher frequency,in particular for lossy samples with a high salt content.We herein present first results obtained on a 1200 MHz spectrometer for a set of biomolecular samples that we have al
23、ready investigated at 850 MHz,and compare sensitivity and resolution in 1H-and 13C-detected NMR spectra.We avoided the temptation to select one“typical”sample,i.e.the very best performing sample that we have,but rather present a selection of samples that we are currently investigating in the laborat
24、ory.We used both,the more classical approach of 13C-detected spectroscopy,which is of advantage when large sample quantities(approx.30 mg)can be prepared,as well as 1H-detected solid-state NMR,which has a mass sensitivity about 50 times higher,and relies on the use of sub milligram protein quantitie
25、s(Lecoq et al.2019;Agarwal et al.2014).Both approaches are today central in biomolecular NMR spectroscopy,and show different strengths and limitations.Proton-detected spectra at 1200 MHz are also under investigation in other labs(Nimerovsky et al.2021).CC-BY-NC-ND 4.0 International licensemade avail
26、able under a(which was not certified by peer review)is the author/funder,who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprintthis version posted March 31,2021.;https:/doi.org/10.1101/2021.03.31.437892doi:bioRxiv preprint 5 Results In th
27、e following,we compare spectra of amyloid fibrils of the fungal prion HET-s(218-289)(Wasmer et al.2008;van Melckebeke et al.2010);sediments of the bacterial helicase DnaB(Gardiennet et al.2012;Wiegand et al.2019);the bacterial RNA helicase and acetyltransferase TmcA(Ikeuchi,Kitahara,and Suzuki 2008;
28、Chimnaronk et al.2009);the Rpo4/7 protein complex of two subunits of archeal RNA polymerase II(Torosyan et al.2019);the filaments of PYRIN domain of mouse ASC(Sborgi et al.2015;Ravotti et al.2016);the viral capsids of the Hepatitis B virus(Lecoq et al.2019)and the African cichlid nackednavirus(Laube
29、r,Seitz at al.2017);supramolecular protein filaments of type 1 pili(Hahn et al.2002;Habenstein et al.2015);and the nonstructural membrane protein 4B(NS4B)of the Hepatitis C virus.In all figures,spectra colored in blue were recorded at 850 MHz,and spectra in red at 1200 MHz.13C-detected 13C-13C corre
30、lation spectroscopy Figure 1a and b compare 13C-and 15N-detected cross-polarization(CP)spectra of HET-s(218-289)amyloid fibrils(Wasmer et al.2008;van Melckebeke et al.2010;Smith et al.2017)measured in a commercial Bruker 3.2mm triple-resonance probe using the E-free design(Gorkov et al.2007)at 850 a
31、nd 1200 MHz.Out of the 71 residues of HET-s(218-289),56 are observed in CP spectra(van Melckebeke et al.2010),the remainder is invisible due to dynamics(Smith 2018;Siemer et al.2006).A significant sensitivity gain is observed in the 13C spectrum at 1200 MHz,although not homogeneous over all resonanc
32、es,but the aliphatic region is favored.We attribute this inhomogeneity of the sensitivity gain to the offset dependence of the CP step caused by the limited rf-field strength available at the 1200 MHz spectrometer on the 13C channel of the probe(the 48 kHz used are not large compared to the 13C spec
33、tral width).The aliphatic regions of 2D 13C-13C Dipolar Assisted Rotational Resonance(DARR)spectra(Takegoshi,Nakamura,and Terao 2001;2003)of HET-s(218-289)recorded at 850 and 1200 MHz are given in Figure 1c,with expanded regions shown in Figure 1d.It can clearly be seen that the spectra at 1200 MHz
34、show higher resolution.However,one can conclude from the 1D traces(Figure S3)that the DARR transfer is,as expected,somewhat less efficient for constant mixing time at the higher magnetic field.Since the MAS frequency of the 1200 MHz 3.2mm probe is currently limited to 20 kHz,corresponding to 66 ppm,
35、some rotational-resonance(Raleigh,Levitt,and Griffin 1988;Colombo,Meier,and Ernst 1988)line-broadening effects are present at 1200 MHz between the carbonyl and aliphatic resonances.The linewidth thus can.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer revi
36、ew)is the author/funder,who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprintthis version posted March 31,2021.;https:/doi.org/10.1101/2021.03.31.437892doi:bioRxiv preprint 6 still be improved by spinning faster;a MAS frequency of around
37、 24 kHz would be optimal,and can generally be achieved in 3.2 mm rotors(Bckmann,Ernst,and Meier 2015).Figure 1:HET-s(218-289)amyloid fibrils.a)Structure model(PDB ID:2RNM)(Wasmer et al.2008)and 1D 13C-detected CP-MAS spectrum,b)1D 15N-detected CP-MAS spectrum,c)20 ms DARR spectra and d)expanded regi
38、ons from the spectra in c.Spectra colored in blue were recorded at 850 MHz and spectra in red were measured at 1200 MHz.CP was matched at 75 and 48 kHz for 1H and 13C at 1200 MHz and 60 and 43 kHz at 850 MHz.Experimental parameters are listed in Table S1.The two spectra were normalized using isolate
39、d well-resolved peaks and the contour levels are the same for the two spectra.As a second system,we show adhesive type 1 pili from E.coli,which assemble in vitro to form long supramolecular protein filaments(Hahn et al.2002;Habenstein et al.2015).Each monomer consists of 150 amino acids.Using 850 MH
40、z data,the 13C resonances have been.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer review)is the author/funder,who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprintthis version posted March 31
41、,2021.;https:/doi.org/10.1101/2021.03.31.437892doi:bioRxiv preprint 7 assigned to 98%of the sequence(Habenstein et al.2015).A clear improvement in resolution at the higher field is observed in the expanded regions shown in Figure2c.While the type 1 pili and HET-s(218-289)were small enough for assign
42、ment and structure determination at 850 MHz(Wasmer et al.2008;van Melckebeke et al.2010)(Habenstein et al.2015),the DnaB helicase from Helicobacter pylori(6 x 59 kDa)with 488 residues per monomer poses a big challenge at 850 MHz,already for assignment.Divide-and-conquer approaches(Wiegand,Gardiennet
43、,Cadalbert,et al.2016;Wiegand,Gardiennet,Ravotti,et al.2016)or segmental isotope labeling of individual protein domains have thus been applied(Wiegand 2018),however without reaching close-to-complete assignment.Figure 3 shows the NMR spectra collected on DnaB complexed with ADP:AlF4-and single-stran
44、ded DNA(Wiegand et al.2019).Figure 3a displays the 13C-detected 1D CP-spectra recorded at three different magnetic field strengths:500 MHz,850 MHz and 1200 MHz.The efficiency of the CP at 1200 MHz suffers again from offset effects,even more than in Figure 1a,as the higher salt content of the sample(
45、130 mM NaCl)reduces the rf-field strengths that can be safely applied according to the manufacturer.Figure 3b shows the 20 ms DARR spectra recorded at these different magnetic field strengths.The increase in resolution with magnetic field is obvious(Figure 3b,c).We have automatically picked the reso
46、nances in the aliphatic region(using CCPNmr(Fogh et al.2002;Vranken et al.2005)of the 2D spectra and find an increase from 203 to 322 peaks between the 850 and 1200 MHz spectra(see Figure S4),highlighting the gain in resolution.This gain will allow us to go further in structural studies of this prot
47、ein using 3D and 4D spectra to further increase resolution.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer review)is the author/funder,who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprintthis
48、version posted March 31,2021.;https:/doi.org/10.1101/2021.03.31.437892doi:bioRxiv preprint 8 Figure 2:Protein filaments of type 1 pili.a)Structural model(PDB ID:2N7H)(Habenstein et al.2015)and 1D 13C-detected CP-MAS spectrum,b)20 ms DARR spectra and c)spectral fingerprints expanded from the spectra
49、in b.Spectra colored in blue were recorded at 850 MHz and spectra in red were measured at 1200 MHz.CP was matched at 70 and 44 kHz for 1H and 13C at 1200 MHz and at 60 and 43 kHz at 850 MHz.CC-BY-NC-ND 4.0 International licensemade available under a(which was not certified by peer review)is the auth
50、or/funder,who has granted bioRxiv a license to display the preprint in perpetuity.It is The copyright holder for this preprintthis version posted March 31,2021.;https:/doi.org/10.1101/2021.03.31.437892doi:bioRxiv preprint 9 Figure 3:The bacterial DnaB helicase.a)1D 13C-detected CP-MAS spectra record