1H / Ultrafast MAS / Paramagnetic
Bernd Reif
Technische Universität München
Helmholtz-Zentrum München
Biomolecular Solid-State NMR Winter School
Stowe, VT
January 10-15, 2016
1. Protons in Solid-State NMR
1) Ultrafast MAS / low power decoupling
2) Application of 1H,1H homonuclear decoupling schemes like
FSLG/PMLG, DUMBO ...
3) Deuteration
Ultrafast MAS
Proton Detection
Heteronuclear Decoupling
1H
line widths of NH3, CH and CH3 in alanine,
measured at 600 MHz
1H
Linewidth [Hz]
MAS=60 kHz
Samoson, Tuherm, Gan, Solid State NMR 20 130 (2001)
Ernst, Samoson, Meier, J. Magn. Res. 163, 332 (2003)
Ernst, Meier, Tuherm, Samoson, Meier, J. Am. Chem. Soc. 126 4764 (2004)
Samoson, Tuherm, Past, Reinhold, Anupold, Heinmaa, Top. Curr. Chem. 246 15 (2005)
Laage, Sachleben, Steuernagel, Pierattelli, Pintacuda, Emsley, J. Magn. Res. 196 133 (2009)
Low-Power XiX Decoupling Schemes at Ultrafast MAS frequencies
low-power (XiX)45:
MAS = 50 kHz
τp = 76 µs
ν1 = 13 kHz
high-power XiX:
MAS = 50 kHz
τp = 57 µs
ν1 = 220 kHz
Gly Cα intensity
high-power XiX:
Optimum for: τp = ca. 2.85 τr
Detken, Hardy, Ernst, Meier, Chem. Phys. Lett. 356 298 (2002)
Ernst, Samoson, Meier, J. Magn. Reson. 203 332 (2003)
Low-Power Sequences for Hartmann-Hahn transfer
n=0 Hartmann-Hahn match condition (ωI = ωS):
Second Order-CP (SOCP)
1H spin-lock efficiency as a function of the applied rf-field
amplitude, indirectly detected by cross polarization to the Cα
resonance of glycine ethyl ester (MAS = 65 kHz)
Lange, Scholz, Manolikas, Ernst, Meier, Chem. Phys. Lett. 468 100 (2009)
Implementation of Low-Power Building-Blocks in
Multidimensional Solid-State NMR experiments
Vijayan, Demers, Biernat, Mandelkow, Becker, Lange ChemPhysChem 10, 2205 (2009)
Frequency Switched Lee-Goldburg (FSLG) and Phase Modulated Lee-Goldburg (PMLG) Experiments
for homonuclear 1H,1H Decoupling
To obtain decoupling,
magnetization has to be rotated around the magic angle in spin space:
PMLG spectrum of a 15N labeled sample of a SH3 domain
PMLG 1H,15N Correlation of the α-spectrin SH3 domain in the solid-state
= ca. 0.24 ppm
@750 MHz
B0 = 750 MHz
rescaled line width
FSLG: Bielecki, Kolbert, Levitt, Chem Phys Lett 155, 341 (1989)
PMLG: Vinogradov, Madhu, Vega, Chem Phys Lett 314 443 (1999)
Protons in Solid-State NMR: Sensitivity and Resolution
3
B03 t
(S /N ) ∝ nγ exc γ Det
u-[2H,15N]-Nac-Val-Leu-OH
€
15N
Detection
1H
Detection
(S /N )[ 1 H ] $ γ H '3 / 2
∝& )
(S /N )[ 15 N ] % γ N (
Sensitivity Gain = x 9.0 @ 33 kHz MAS
≈ (10)
JMR 151, 320-327 (2001); JMR 160, 78-83 (2003)
€
3/2
= 30
Proton density in the deuterated α-spectrin SH3 domain
fully protonated
perdeuterated with 100% of labile protons
back-exchanged
500 MHz, 10 kHz MAS
Chevelkov et al. J. Am. Chem. Soc. 125, 7788 (2003)
Proton density in the deuterated α-spectrin SH3 domain
perdeuterated with 10% of labile protons
back-exchanged
Chevelkov et al. Angew. Chem. Int. Edt. 45 3878-3881 (2006)
Proton dilution in small organic molecules
1% protonated Ala
(= 99% deuterated)
MAS = 11 kHz
B0 = 360 MHz
MREV-8 CRAMPS
spectrum
1H
McDermott, Creuzet, Kolbert, Griffin,
J. Magn. Reson. 98, 408 (1992)
1H
Zheng, Fishbein, Griffin, Herzfeld,
J. Am. Chem. Soc. 115, 6254 (1993)
Deuteration in Solid-State NMR
Paulson, Morcombe, Gaponenko, Dancheck, Byrd & Zilm, Sensitive High Resolution Inverse
Detection NMR Spectroscopy of Proteins in the Solid State. J Am Chem Soc 125: 15831-15836
(2003)
Morcombe, Gaponenko, Byrd & Zilm, C-13 CP MAS spectroscopy of deuterated proteins: CP
dynamics, line shapes, and T-1 relaxation. J Am Chem Soc 127: 397-404 (2005).
Zhou & Rienstra, High-Performance Solvent Suppression for Proton-Detected Solid-State NMR. J
Magn Reson 192: 167–172 (2008).
Zhou, Shah, Cormos, Mullen, Sandoz & Rienstra, Proton-detected solid-state NMR Spectroscopy of
fully protonated proteins at 40 kHz magic-angle spinning. J Am Chem Soc 129: 11791-11801
(2007).
Schanda, Huber, Verel, Ernst & Meier Direct Detection of 3hJNC Hydrogen-Bond Scalar Couplings in
Proteins by Solid-State NMR Spectroscopy. Angew Chem Int Edt 48: 9322-9325 (2009)
Schanda, Meier, Ernst Quantitative Analysis of Protein Backbone Dynamics in Microcrystalline
Ubiquitin by Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 132: 15957–15967 (2010).
Solvent Suppression in the Solid-State
a,b: zg
c: x-filter
d: x-filter + gradients
Chevelkov et al.
JACS 125 7788 (2003)
Solvent Suppression in the Solid-State
Solution 2:
Zilm and co-worker JACS 125 15831 (2003)
Solution 3: MISSISSIPPI
Rienstra and co-worker JMR 192 167 (2008)
High power RF irradiation
Imagine what happens to your sample (40 µL) when you apply 100 W for 30 ms ...
High power RF irradiation
Imagine what happens to your sample (40 µL) when you apply 100 W for 30 ms ...
Using the water chemical shift as a thermometer
Sample Heating due to Friction (3.2 mm rotor)
"Static" Sample heating due to RF irradiation
Total time of irradiation d1 = 30 ms
ωrf = 100 kHz, 100 mM NaCl
recycle delay = 3s
regular CP-MAS probe
a: no irradiation
b: d2 = 2.7 s
c: d2 = 1.7 s
d: d2 = 1.0 s
e: d2 = 0.03 s
Static/Steady-state sample heating due to
RF irradiation (cw = 30 ms)
"Dynamic" Sample heating due to RF irradiation
Total time of irradiation d1 = 30 ms
ωrf = 100 kHz, 100 mM NaCl
recycle delay = 3s
d5 = 1 ms (switching delay)
regular CP-MAS probe
a: no irradiation
b: t1 = 0 ms
c: t1 = 10 ms
d: t1 = 20 ms
e: t1 = 30 ms
Dynamic sample heating in an indirect evolution period
due to RF irradiation
d3 = 3s
What can you do with protons in the solid-state ?
Solution-State like Pulse Schemes applied to Crystalline Proteins
HNCACB
Coherences are sufficiently long-lived in the solid-state to enable scalar transfers combined with 1H detection
Increased reliability in the assignment of resonances in MAS solid-state NMR
Linser, Fink, Reif J. Magn. Reson. 193, 89 (2008)
Linser, Fink, Reif, J. Biomol. NMR 47, 1 (2010)
More assignment experiments
Linser, R., Fink, U., and Reif, B. (2010). Narrow carbonyl resonances in proton-diluted proteins
facilitate NMR assignments in the solid-state. J Biomol NMR 47, 1-6.
Linser, R. (2011). Side-chain to backbone correlations from solid-state NMR of perdeuterated
proteins through combined excitation and long-range magnetization transfers. J Biomol NMR 51,
221-226.
Linser, R. (2012). Backbone assignment of perdeuterated proteins using long-range H/C-dipolar
transfers. J Biomol NMR 52, 151-158.
Barbet-Massin, E., Pell, A.J., Jaudzems, K., Franks, W.T., Retel, J.S., Kotelovica, S., Akopjana, I.,
Tars, K., Emsley, L., Oschkinat, H., Lesage, A., and Pintacuda, G. (2013). Out-and-back C-13-C-13
scalar transfers in protein resonance assignment by proton-detected solid-state NMR under ultra-fast
MAS. J Biomol NMR 56, 379-386.
Barbet-Massin, E., Pell, A.J., Retel, J.S., Andreas, L.B., Jaudzems, K., Franks, W.T., Nieuwkoop,
A.J., Hiller, M., Hagman, V., Guerry, P., Bertarello, A., Knight, M.J., Felletti, M., Le Marchand, T.,
Kotelovica, S., Akopjana, I., Tars, K., Stoppini, M., Bellotti, V., Bolognesi, M., Ricagno, S., Chou, J.J.,
Griffin, R.G., Oschkinat, H., Lesage, A., Emsley, L., Herrmann, T., and Pintacuda, G. (2014). Rapid
Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning. J Am Chem Soc 136,
12489-12497.
Chevelkov, V., Habenstein, B., Loquet, A., Giller, K., Becker, S., and Lange, A. (2014). Protondetected MAS NMR experiments based on dipolar transfers for backbone assignment of highly
deuterated proteins. J Magn Reson 242, 180-188.
Residual Protonation in Perdeuterated Proteins
HANAH (1H Natural Abundance in 2H Proteins)
Residual Protonation in perdeuterated proteins
(> 97% 2H, 99% 13C -Glucose)
10% labeling of -CD2H
α-spectrin SH3
Methyl 1H,13C Correlations at 1H „Natural Abundance“
1H,13C
1H,13C
double CP
HMQC
Agarwal et al. JMR 194 16 (2008)
Increased sensitivity using specific precursors for amino acid biosynthesis
α-spectrin SH3
α-ketoisovalerate:
Sparse labeling at Leu, Val sites
(no measurable 13C-13C spin diffusion)
H
D
C
D
Goto, Gardner, Mueller, Willis, Kay, JBNMR 13, 369 (1999)
Agarwal, Diehl, Skrynnikov, Reif, JACS 128, 12620 (2006)
Agarwal, Xue, Reif, Skrynnikov, JACS 130, 16611 (2008)
Detection of Exchangeable Hydroxyl Protons
α-spectrin SH3
W41ε
?
Exchangeable Hydroxyl Protons in α-spectrin SH3
13C
detected 1H,13C-CP
∼ 1 ms
HN
C´
Cα
Exchangeable Hydroxyl Protons in α-spectrin SH3
Exchangeable Hydroxyl Protons in α-spectrin SH3
13C
Agarwal et al. JACS 132 3187 (2010)
detected 1H,13C-CP
Exchange Properties of Hydroxyl Protons (α-SH3, 5°C)
Hydrogen Bonds in α-spectrin SH3: Y15(OH)-E22(COO-)
Hydrogen Bonds in α-spectrin SH3: Y15(OH)-E22(COO-)
Hydrogen Bonds in α-spectrin SH3: T24(OH)-E17(COO-)
0.94
%r
(3
Int C_O_H
DC _ O _ H
H ...O=C
= '
*
(τ mix → ∞) = D
Int H...O= C
& rC _ O _ H )
H ...O=C
DH , C = −µ0
0.53
€
γ H γC !
rH3 , C
€r(OH) = ca. 1.08-1.10 Å
4D Solid-State NMR Experiment for Structure Determination
Huber et al. Meier Chem Phys. Chem. 12, 915-918 (2011)
Use of time shared evolution periods in 3D/4D experiments
2D F3/F4 plane (red) of the 4D HN…NH
Linser et al. JACS 133, 5905-5912 (2011)
High Resolution Deuterium MAS solid-state NMR spectroscopy
1) Analysis of Side Chain and Backbone Dynamics
2) In Solution-State, 2H resonances are broad due to the J(0) contribution to 2H T2
CQ = ca. 55 kHz (methyl) and 165 kHz
In solution:
2
2
1 " e qQ %
R(D+ ) = $
' [9J(0) + 15J(ω D ) + 6J(2ω D )]
80 # ! &
τf
1
1
τR
J(ω ) = S 2
+ (1− S 2 )
2
9 1+ (ωτ R )
9
1+ (ωτ f ) 2
In solids
€
Deuterium Single Quantum (SQ) Correlations
Setup-System:
2H,13C,15N-NAc-Val-Leu-OH
Deuterium Double Quantum (DQ) Correlations
DQ chemical shifts are scaled by 1/2
Setup-System:
2H,13C,15N-NAc-Val-Leu-OH
Making use of the spin-1 properties of 2H:
HQ =
ωQ
[3Iz Iz − I ⋅ I]
3
€
τ = 3 µs (black), 9 µs (red/blue)
> Factor 2 increased resolution in the DQ experiment
Where does the additional increase in resolution (> 2) come from?
ωQ
[3Iz Iz − I ⋅ I]
3
e 2qQ 1
2
ωQ =
[
2 (3cos θ −1)] ;
2I(2I −1)
HQ =
η=0
€
2H-DQ
are insensitive to MA mis-setting
and to Motional Broadening
See Vega, Pines (1977) J. Chem. Phys. 66 5624
Simulated 2H off-Magic Angle spectra
CQ = 100 kHz
MAS = 20 kHz
Setting the Magic Angle using NaNO3
2H
13C
spectroscopy applied to α-spectrin SH3
detected 1H,13C CP
2H-DQ,13C
CP
Dmet-Cmet
Dα-Cα
2H
spectroscopy applied to α-spectrin SH3: Methyl spectral region
13C
detected 1H,13C CP
2H-DQ,13C
CP
S/N(2H,13C) ≈ 2-3 x S/N(1H,13C)
γ(1H) = 6.5 x γ(2H)
T1 (1H) = 4 x T1 (2H)
n(2H) = 33-36 x n(1H)
2H
spectroscopy applied to α-spectrin SH3
Cα-Dα
Agarwal, Faelber, Schmieder, Reif, JACS 131, 2 (2009)
Can we detect aliphatic protons ?
(other than methyls)
Can we detect aliphatic protons (other than methyls) ?
Biosynthesis with 2H,13C glucose and various amounts of H2O (5-30 %)
NOESY type distance restraints in the solid-state
3. Paramagnetic
Loss:
Dilution: x10
Gain:
Line width: x4
Proton detection: x9
How can we do better ?
Doping with Cu(II)-EDTA
0 mM Cu-EDTA
75 mM Cu-EDTA
Wickramasinghe, Kotecha, Samoson, Past, Ishii J. Magn. Reson. 184, 350 (2007)
Linser, Diehl, Chevelkov, Reif J. Magn. Reson. 189, 209 (2007)
PREs are not uniform. Accessibility of an Amide Proton in the SH3 domain of α-spectrin
- Cu(II)EDTA
+ 75 mM Cu(II)EDTA
Is the differential 1H-T1 due to a variation of the HN distance to the surface of the protein ?
Surface Distance of an Amide Proton in α-spectrin SH3
ΔR1(1H): R1(with Cu-EDTA) – R1(without Cu-EDTA)
Disentangling Paramagnetic Effects from Intrinsic Differences in 1H-T1 Relaxation
ΔR1(1H) is a Function of the Surface Distance of an Amide Proton
ΔR1 = k /r 6
2 2
2
0
$ µ '2 2γ n ge µB
3τ c
6τ c
τc
Se ( Se + 1) & 0 ) /
+
+
2 2
2 22
2 2
% 4 π ( .1+ (ω n − ω e ) τ c 1+ ω n τ c 1+ (ω n + ω e ) τ c 1
15
1/ τ c = 1/ τ e + 1/ τ n
k=
€
€
€
Integration over the
solvent accessible volume
yields:
r$ max 2 π θ max
ΔR1, eff = k ⋅ reff−6
reff−6 =
∫ ∫ ∫r
0
0
−6
⋅ r$2 sin θ dθ dϕ dr$
r = r02 + r"2 + 2r0 r"cos θ
0
Solomon, Phys. Rev. 99, 559 (1955)
€
€
€
ΔR1(1H) is a Function of the Surface Accessibilty of an Amide Proton
dashed line:
θmax = 60° and 120°
solid line:
θmax = 90°
150 mM, 0 mM Cu-EDTA
r = 4.0 Å
Linser et al. J. Am. Chem. Soc. 131 1307 (2009)
Faster is better ….
u-[2H,15N]-GB1, 39 kHz MAS, 750 MHz: Back-exchanged with 100 % H2O
Zhou, Shea, Nieuwkoop, Franks, Wylie, Mullen, Sandoz, Rienstra (2007) Angewandte Chemie Int. Edt. 46 8380-8383
u-[2H,15N] α-SH3
re-crystallized with 100 % H2O
60 kHz MAS, 1000 MHz
Lewandowski, Dumez, Akbey, Lange, Emsley, Oschkinat (2011) J. Phys. Chem. Lett. 2 2205-2211
1.3 mm rotors yield approximately the same absolute intensity as fully packed 3.2 mm rotors
2H,15N
α-SH3 as a function of [H2O]
(during crystallization)
Akbey et al., JBNMR 46, 67-73 (2010)
MAS
rotor
Volume
max concentration
exchangeable protons
relative
intensity
1.3 mm
4 µL
100 %
0.5
3.2 mm
40 µL
ca. 20 %
1
Proton detection at FAST MAS with Protonated Protein Samples
Protonated GB1, u-[1H,15N], 39 kHz MAS
Zhou, Shah, Cormos, Mullen, Sandoz, Rienstra (2007) J. Am. Chem. Soc. 129 11791-11801
Proton detected experiments of Protonated Protein Samples
800 MHz, MAS = 60 kHz
ε186 subunit from DNA polymerase III
tetrameric single-stranded DNA binding protein (SSB)
Marchetti, Jehle, Felletti, Knight, Wang, Xu, Park, Otting, Lesage, Emsley, Dixon, Pintacuda (2012)
Angewandte Chemie Int. Edt. 51 10756-10759
Proton detected experiments of Protonated and Deuterated Protein Samples
u-[1H,15N] SSB, 800 MHz
Marchetti et al. (2012)
Angewandte Chemie Int. Edt.
51, 10756-10759
u-[2H,15N] α-SH3, 1000 MHz
Lewandowski et al. (2011)
J. Phys. Chem. Lett. 2 2205-2211
when fast is not fast enough …
Ubiquitin, u-2H,13C,15N, recrystallized from 100 % H2O
MAS: 100 kHz, 850 MHz
Agarwal et al. Meier Angewandte Chemie Int. Edt. 53, 12253-12256 (2014)
Applications to non-crystalline systems
Soluble Protein Complexes: The proteasome activator
complex 11S-α7(β7β7)α7 [Thermoplasma acidophilum]
perdeuterated, back-exchanged with 20% H2O; 20 kHz MAS, 600 MHz
11S-α7β7β7α7
1.1 MDa
Mainz et al., Angewandte Chemie Int. Edt. 52 8746-8751 (2013)
MAS induces reversible sedimentation
of protein complexes
αB inhibits
amorphous
aggregation of reduced lysozyme
Challenges in the Solid-State:
- Crystal contacts
- Ligands have to be co-precipitated
- Analysis of chemical shifts is ambiguous if crystal symmetry changes
Challenges in Solution-State:
- Correlation time problem: Resonance Lines become broad for large
molecules
- TROSY (Pervushin/Wüthrich)
- Decrease of τC: High temperature / Reversed Micelles (Wand)
Bertini et al., PNAS 108, 10396 (2011)
Ribosomal Complexes: How far can we go ?
Questions:
• Can we observe individual components of the ribosome ?
• Does ribosome binding induce a conformational change of Trigger Factor (TF) ?
in collaboration with
Shang-Te Danny Hsu, Taiwan
and Roland Beckmann, LMU München
Barbet-Massin, Angewandte Chemie Int. Edt. 54, 4367 (2015)
TF-RBD in complex with the 50S ribosome
in complex with 50S
TF-RBD in complex with 50S
solution-state (TF-RBD)
unbound TF-RBD in solution
TF-RBD in complex with the 50S ribosome
no chemical shift changes
chemical shift changes /
exchange broadening
solution-state (TF-RBD)
in complex with 50S
TF (Ribosome Binding Domain) (~14 kDa)
in complex with 50S (~1.4 MDa)
100 % back-exchanged @ 60 kHz MAS:
ca. 20 µg TF-RBD
Baram et al. Yonath PNAS 102 12017 (2005)
Processing of the Amyloid Precursor Protein (APP)
yields the Alzheimer's disease β-amyloid peptide Aβ
Solid-State NMR of Aβ aggregates
Lansbury et al. Griffin, Nat. Struct.Biol. 2, 990 (1995)
Benzinger et al. Meredith, Proc. Natl. Acad. Sci. USA 95, 13407 (1998)
Petkova et al. Tycko, Science 307, 262 (2005)
Petkova et al. Tycko, Biochemistry 45, 498 (2006)
Paravastu, et al. Tycko, Proc. Natl Acad. Sci. USA 106, 7443 (2009)
Bertini et al. Mao J. Am. Chem. Soc. 133, 16013 (2011)
Chimon et al. Ishii, Nature Stuct. Mol. Biol. 14, 1157 (2007)
Ahmed et al. Smith, Nature Struct. Mol. Biol. 17 561 (2010)
Amyloids: Alzheimer’s disease Aβ40 fibrils
50 nm
Tex
• 1 set of resonances (30-40 peaks)
• Well defined chemical shift dispersion
→ well-defined 3D structure
3D-HNCO
Linser et al., Angewandte Chem. Int. Edt. 50, 4508-4512 (2011)
Alzheimer's disease Amyloid Aβ(1-40)
1H,15N CP correlation (full spectrum)
Lys-28 ?
Histidine imidazole
correlations ?
Alzheimer's disease Amyloid Aβ(1-40)
1H,13C CP correlation
Ser
H 2O
His
Detection of Histidines Imidazole Protons in Amyloid Aβ(1-40) fibrils
Detection of Histidines Imidazole Protons in Amyloid Aβ(1-40) fibrils
-
Hδ1
Detection of Histidines Imidazole Protons in Alzheimer's disease Amyloid Aβ(1-40) fibrils
H13
Q15
E11
H14
K16
E22
Tycko, Quart Rev Biophys. 39 1-55 (2006)
Hδ1
Cα
Gln-Cβ/
Glu-Cβ
Exchangeable side chains
can assist in determining
the quarternary structure
of amyloid fibrils
Histidines can assist in determining the quarternary
structure of Amyloid Aβ(1-40) fibrils
Hδ1
Agarwal et al., PCCP 15, 12551 (2013);
see also: Petkova et al. PNAS 99 16742 (2002)
Membrane proteins: OmpG and Bacteriorhodopsin
Bacteriorhodopsin
OmpG
Linser et al. Angewandte Chemie Int. Edt. 50, 4508-4512 (2011)
Protein-RNA Complexes: The boxCD - L7ae complex
of Archeoglobus fulgidus
solution-state
solid-state
Asami et al. Angewandte Chemie Int. Edt. 52 2345-2349 (2013)
HN correlations of the boxCD RNA-Protein Complex
Superposition of the Solid-State and
Solution-State Spectra
solution-state
solid-state
Protein HN correlation spectra
in presence of protonated and deuterated RNA
Other examples: Proton detection in noncrystalline systems
Ward, M.E., Shi, L., Lake, E., Krishnamurthy, S., Hutchins, H., Brown, L.S., and Ladizhansky, V.
(2011). Proton-Detected Solid-State NMR Reveals Intramembrane Polar Networks in a Seven-Helical
Transmembrane Protein Proteorhodopsin. J Am Chem Soc 133, 17434-17443.
Andreas, L.B., Reese, M., Eddy, M.T., Gelev, V., Ni, Q.Z., Miller, E.A., Emsley, L., Pintacuda, G.,
Chou, J.J., and Griffin, R.G. (2015). Structure and Mechanism of the Influenza A M2(18-60) Dimer of
Dimers. J Am Chem Soc 137, 14877–14886.
Chevelkov, V., Habenstein, B., Loquet, A., Giller, K., Becker, S., and Lange, A. (2014). Protondetected MAS NMR experiments based on dipolar transfers for backbone assignment of highly
deuterated proteins. J Magn Reson 242, 180-188.
Acknowledgement
Vipin Agarwal
Sam Asami
Veniamin Chevelkov
Rasmus Linser
Purdue University
Nikolai Skrynnikov