EPR in Structural Biology - PDF Free Download

EPR in Structural Biology Peter Höfer Product Manager EPR Pittsburgh April 2016 Innovation with Integrity

EPR species naturally occurring Metal Centers: Cu 2+, Mn 2+, Fe 3+, Mo 5+, Radicals: tyrosine, tryptophan, quinol, carotenoid Antioxidants: ascorbate, polyphenols, nitroaromatic drugs Small Molecule: NO, H 2 O 2, O 2, OH Defect Centers: O-vacancy, irradiation damage

EPR species reporter molecules Spin Probes: TEMPOL, Trityl, DPPH Not binding Spin Labels: PROXYL, MTSSL Binding SO 2 CH 3 S Protein H 3 C H 3 C N S CH 3 CH 3 + HS C Protein H 3 C H 3 C N S CH 3 CH 3 Cys C O Cys O Spin Traps: DMPO, DEPMPO, PBN, MNP; CMH Transforming

EPR samples Liquids and Solids Can be measured on the same instrument (same probehead) For low temperature VT accessories are required. T range 4-300K: He or cryogen-free VT, 100 500K: liq. and gaseous N 2 Sample concentration range and typical volume X-band: nm-m & 50-150 µl For aqueous sample @ RT capillaries or flat cells are used instead of tubes Q-band: nm-mm & 5 15 µl No molecular size restriction! In-vitro and in-vivo

Spin Labelling Bio Molecules (Most) proteins and nucleic acids don t have unpaired electrons! no EPR?!... but... we can introduce the wanted unpaired electron into almost any system under investigation and we can do this also (almost) wherever we want Site directed spin labeling (SDSL) 4/18/2016 5

What Is a Spin Label? a stable chemical compound which possesses an unpaired electron (i.e. it is a stable radical) and a specific reactive group which binds to (bio)molecules. The vast majority of spin labels are nitroxides, where the unpaired electron is located at an NO group, which is usually part of a heterocyclic ring. Functional groups contained within the spin label allow them to be attached to the molecule under investigation and determine their specificity. e.g.: Protein thiol groups (SH C) specifically react with the functional groups of the spin label methanethiosulfonate, maleimide, and iodoacetamide, creating a covalent bond with cysteine. Any amino acid of the protein, one at the time, can be replaced with cysteine by site directed mutagenesis. If a natural Cys present outside of the region of interestadditional site directed mutagenesis step (Cys to Ser) is required, unless the native Cys is buried and hence not accessible to the spin label. A little bit of history: Spin labels were first synthesized in the laboratory of H. M. McConnel in 1965. The first spin labelling studies have been performed using thiol specific functional groups to label natively occurring cysteins in proteins (e.g. in hemoglobin). Site directed spin labeling (SDSL) was pioneered in the laboratory of Dr. W. L. Hubbell in the late 80s/early 90s. 4/18/2016 6

SDSL-EPR-Tools: Overview Spin Label Mobility reflected in the EPR spectral lineshapes => provide a fingerprint of the protein fold and its dynamics Accessibility towards paramagnetic relaxation enhancement molecules (NiEDDA, CrOx, O 2 ) => Discrimination between lipid bilayer, aqueous phase and protein interior Polarity of the SL microenvironment => reflected in the A zz. Increasing A zz points to shifts in polar environment Spin Spin Distance determination (~ 8 100 A) CW EPR: 8 20 A DEER: 15 100 A Information about Structure and Dynamics 4/18/2016 7

Spin Label Environment Effects from molecular motion N Effects from relaxation N Free tumbling 30 um Moderately immobilized 15 mm Strongly immobilized 50 mm Characterize the paramagnetic center environment

Spin Label Mobility EPR-SDSL & Intrinsically Disordered Proteins EPR spectral shape broadening in the case of a disorder to order transition due to an induced folding mechanism Before After folding EPR spectral shape is sensitive to the mobility of the label which is described by the rotational correlation time ( c ) A spectral modification represents change in the environment of the label affecting its mobility and thus reveals structural transitions such as folding events Region of the intrinsically disordered N TAIL C-terminal domain that undergoes an α-helical-induced folding in the presence of the partner protein P XD.

Solvent Accessibility spectrum saturation bound free Lactate Dehydrogenase (LDH) NAD + crystal structure. Calculated solvent accessible surface. Spin Label attachment at 1 24 mw 23.9 mw 4 mw 3.9 mw In collaboration with W. Trommer bound 11 mw 11.1 mw 0.1 0.01 0.1 1 10 100 P / mw P 1/2 P 1/2 P 1/2 free 1.8 mw 2 mw 0.01 0.1 1 10 100 P / mw P 1/2 in air in nitrogen

Polarity of the SL microenvironment a 0 N Dependence of the isotropic hyperfine coupling, a 0N, on solvent polarity, for DOXYL (circles) and TOAC (squares) spin labels in protic solvents April 18, 2016 11

EPR Product Portfolio CW-EPR EMXnano, EMXmicro, EMXplus Multi purpose research instruments Innovation with Integrity

Distance measurements Electron Spin as a Molecular Microscope Pulse-EPR: DEER/PELDOR S-S: 15 100 Å Pulse-EPR: ESEEM, HYSCORE, ENDOR CW-ENDOR S-I: <8Å CW-EPR S-S: 5-20 Å S-I: 0-4 Å

Electron-Nuclear Hyperfine Coupling isotropic anisotropic

Pulse EPR: ESEEM Lipid Membrane /2 #D 2 O seen by SL at the membrane surface D 2 O solvent 3p-ESEEM #D2O 2p-ESEEM

Electron-Electron Dipolar Coupling E pump observe D r -3 r / nm dd / MHz 1.5 15.4 2.0 6.5 2.5 3.3 3.0 1.9 dd 2 = dd = 0 g 1 g 2 2 e ( 3 cos 2 1 ) 2 h r 3

Pulse EPR: Dipolar Spectroscopy DEER/PELDOR Dipolar oscillation observe pump Background 3420 3450 3480 3510 Field / G 3530 /2

Pulse EPR: Dipolar EPR Spectroscopy DEER/PELDOR Pulsed Double Electron Electron Resonance (DEER) Spectroscopy: Measures the dipole dipole interaction between two unpaired electron spins and is being used to determine long range distances (15-100 Å) A O N R AB 1 B O N Normalized Echo Amplitude 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1 2 3 4 5 6 Distance [nm] 0 1000 2000 3000 4000 5000 t [ns]

DEER vs other techniques DEER covers the entire length scale Innovation with Integrity

Pulsed EPR: Dipolar EPR Spectroscopy DEER/PELDOR Wild Type SH- OH- site-directed mutagenesis Cys spin labeling with MTSSL DEER Experiment & Analysis Normalized Echo Amplitude 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1 2 3 4 5 6 Distance [nm] 0 1000 2000 3000 4000 5000 t [ns] Information content Distance range: 15-100 Å Distance distribution Orientation information Correlate structure and structural changes to functionality

Pulsed EPR: Dipolar EPR Spectroscopy DEER/PELDOR Samples: proteins, RNA, DNA, protein-protein, protein-rna complexes Spin labels Types of paramagnetic centers Radicals endogenous: tyrosine tryptophan, quinone exogenous: nitroxide and trityl spin labels Transition ion metals endogenous: Cu, Fe, Mo/W, Ni, Co, Mn exogenous: Mn, Cu, Gd, spin labels Typical concentration 50-200 µm Volume X-Band: 50-100 µl Q-Band: 5 15 µl Temperature: typically 50K-100K Advantages: No limitations on molecular size Works in phospholipids Works in-cell

Pulsed: Dipolar EPR Spectroscopy PELDOR / DEER: Proteins The distance between a single pair of spin labeled mutants is measured at a time Distance determination between multiple spin labels is possible however the analysis is more complicated

Pulsed: Dipolar EPR Spectroscopy PELDOR / DEER: Membrane Proteins Black=open, red=closed Distance determination in various intermediate states direct observation of large conformational changes Distance measurements in liposomes Explore structure and conformational dynamics in native-like environment

Pulsed EPR: Dipolar EPR Spectroscopy PELDOR / DEER: RNA & DNA In-Vivo In-cell: RNA and DNA Strong change in vitro vs in cell In-vivo determination of intramolecular distances in nucleic acids understanding their conformational flexibility

Pulsed EPR: Dipolar EPR Spectroscopy PELDOR / DEER: NMR Meets EPR Binding of RsmE protein to the RsmZ srna: Combining NMR and EPR: Powerful, novel approach for structure determination of large protein RNA complexes

ELEXYS E580: DEER/PELDOR Pulse EPR ELEXYS E580 Q-Band ~ 25 min Acquisition time X-Band ~ 22 h Acquisition time Dedicated GUI for ease of use Recent S/N improvements: Going from X- to Q-Band (> 50) Shaped pulse (> 3) Gain in throughput!

References Spin Label Mobility (slide 9) Martinho, M., et. al., Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus necleoproteins by site-directed spin labeling EPR spectroscopy (2012) 13(5), p453. doi: 10.1080/07391102.2012.706068 Polarity of Microenvironemnt (slide 11) Marsh, D., Spin-Label EPR for Determining Polarity and Proticity in Biomolecular Assemblies: Transmembrane Profiles, Appl Magn Reson (2010) 37(1-4), p435. doi:10.1007/s00723-009- 0078-3 DEER (slide 22) Lumme, C., et. al., Nucleoties and Substrates Trigger the Dynamics of the Toc34 GTPase Homodimer Involved in Chloroplast Preprotein Translocation, Structure (2014), http://dx.doi.org/10.1016/j.str.2014.02.004 DEER (slide 23) Duerr, K. L., et. al., Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states. Cell (2014) 158(4), p 778. doi: 10.1016/j.cell.2014.07.023 Zou, P., et. al., Conformation Cycle of the ABC transporter MsbA in Liposomes. Detailed Analysis using Double Electron-Electron Resonance Spectroscopy, J Mol. Biol. (2009) 393(3), p586. doi:10.1016/j.jmb.2009.08.050 Mchaourab, H. S., et. al., Toward the Fourth Dimension of Membrand Protein Structure: Insight into Dynamics from Spin0labeling EPR Spectrscopy, Structure (2011) 19(11), p 1549. doi:10.1016/j.str.2011.10.009 Copyright 2012 Bruker Corporation.

References DEER (slide 24) Krstic, I., et. al., Long-Range Distance Measurements on Nucleic Acids in Cells by Pulsed EPR Spectroscopy. Angew. Chem. Int. Ed. (2011) 50(22), p 5070. doi: 10.1002/anie.201100886 Haensel, R., et. al., In-Cell NMR and EPR Spectroscopy of Biomacromolecules, Angew. Chem. Int. Ed. (2014) 53(39), p10300. doi: 10.1002/anie.201311320 DEER (slide 25) Duss, O., et. al., EPR-aided approach for solution structure determination of large RNAs or protein-rna complexes, Nature Comm. (2014) 5. doi: 10.1038/ncomms4669 Duss, O., et. al., Structural basis of the non-coding RNA RsmZ acting as a protein sponge, Nature (2014) 509, p588. doi: 10.1038/nature13271 Copyright 2012 Bruker Corporation.