The Bradford Assay with DMGDH samples in 3 M Gu/HCl (30 mM GuHCl in assay – constant) is sensitive to the order of addition of reagents (maybe due to protein precipitation in assay format A?). Assay method B Bradford reagent 1 ml 0.1 M NaCl : 90 ul 3 M Gu/HCl buffer (10 – x) ul Protein solution x ul Assay method A 0.1 M NaCl : 90 ul 3 M Gu/HCl buffer (10 – x) ul Protein solution x ul Bradford reagent 1 ml From MariangelaCamozzi thesis No such problem with BCA assay
Theoretical/calculated 280 value for a protein http://web.expasy.org/protparam/ The principle: 280 = n Tyr* 280,Tyr + n Trp * 280,Trp + n cystine * 280, cystine Where: 280,Tyr = 1490 M -1 cm -1 , 280,Trp = 5500 M -1 cm -1 280,cystine = 125 M -1 cm -1 calculated at pH 6.5, in 6.0 M guanidinium hydrochloride, 0.02 M phosphate buffer. Two values: one assuming that all Cys are free (no Abs), one assuming that all Cys form SS bonds (low 280,cystine ) Gu/HCl should have little effect on 280 , but better check with denatured and dialyzed protein
If your protein contains a chromophor absorbing light in the UV region....
Take into account the presence of the chromophore, which absorbs light in the UV, to calculate the 280 of the protein Example: Dimethylglycine dehydrogenase (DMGDH), a mitochondrial enzyme containing covalently bound FAD, participating in the metabolism of one-carbon units 280 = n Tyr* 280,Tyr + n Trp * 280,Trp + n cystine * 280, cystine + n FAD * 280, FAD from the protein sequence and 1 for holo-DMGDH http://web.expasy.org/protparam/ determined experimentally in buffer + GuHCl
Submit DMGDH sequence to http://web.expasy.org/protparam/ Output: Number of amino acids: 861 Molecular weight: 96236.7 Theoretical pI: 6.74 Two (not too different) values of the 280 : 280 = 143505 M -1 cm -1 assuming all pairs of Cys residues form cystines (Abs 0.1% (=1 g/l) = 1.491) 280 = 143130 M -1 cm -1 assuming all Cys residues are reduced (Abs 0.1% (=1 g/l) = 1.487)
Determine 280, FAD experimentally in buffer + GuHCl using known at 450 nm of FAD in diluted buffer without GuHCl, pH 7. 264 nm 267 nm 280 nm 448 nm Buffer + GuHCl buffer 448, FAD 11.9 mM ‐1 cm ‐1 11.3 mM ‐1 cm ‐1 280, FAD 19.95 mM ‐1 cm ‐1 24.32 mM ‐1 cm ‐1
Calculation of protein extinction coefficient taking into account the bound cofactor/coenzyme: use 280 of protein (from Protparam) AND 280 of coenzyme in guanidine (in mM -1 cm -1 ); 280‐protein 280‐FAD 280‐EFAD 448‐EFAD A280/A448 f(holo) 143.13 24.32 167.45 11.9 14.0714286 1 143.13 21.888 165.018 10.71 15.4078431 0.9 143.13 19.456 162.586 9.52 17.0783613 0.8 143.13 17.024 160.154 8.33 19.2261705 0.7 143.13 14.592 157.722 7.14 22.089916 0.6 + Apo‐DMGDH Holo‐DMGDH The A 280 /A 448 ratio of the denatured protein solution can be used to determine the fraction of holoEnzyme in the DMGDH prep.
Combine absorption and fluorescence spectroscopies to identify the coenzyme bound to the protein
MICAL, the novel multidomain flavoenzyme participating (not only) in actin cytoskeleton dynamics Flavin ox Flavin red R R N O N N O N NAD(P)H N N N N O O H SOH + H 2 O O 2 S (monooxygenaseactivity) R O N N H 2 O 2 Flavin ox + N (oxidaseactivity) N O O H OH Flavin ox + O 2.‐ 4a‐hydroperoxy‐Flavin
Use of absorption in the visible region to identify, characterize and quantify the protein 0.14 MICAL-His 0.12 MICAL-His + 0.2% SDS 0.10 ε 457 = 8.0 mM -1 cm -1 Absorbance 0.08 0.06 0.04 0.02 0.00 300 400 500 600 700 800 Wavelength, nm Absorption spectrum of the purified N-terminal domain of h-MICAL • The spectrum of the «as isolated» MICAL indicates the presence of a flavin coenzyme forming a charge-transfer complex (with Trp400 from X-ray structure) • Release of the coenzyme by denaturation yields the spectrum of free flavin (to be identified as FAD or FMN) If FAD from the known 448 of free FAD (11.3 mM -1 cm -1 ) we can calculate • the 458 of MICAL-bound FAD as 8.1 mM -1 cm -1 If FMN the known 446 of free FMN (12.2 mM -1 cm -1 ) should be used •
Excitation and emission spectra of flavin coenzymes Emission spectrum, λ ex : 448 nm Excitation spectrum, λ em : 523 nm • The excitation spectrum reflects the absorption spectrum. • The shape of the emission spectrum is independent from λ ex • By comparing excitation and emission spectra, the compound may be identified
Fluorimetric Indentification of the cofactor bound to MICAL Fp domain as FAD exploiting the different fluorescence of FAD and FMN due to quenching of the isoalloxazine fluorescence by the AMP moiety phosphodiesterase + Mg ++ AMP FMN FAD 40 λ ex , 450 nm 35 30 Fluorescence 25 + PDE 20 15 10 free flavin coenzyme 5 0 500 520 540 560 580 600 Wavelength, nm Native If FAD, PDE will bring along a 10x increase of fluorescence due to conversion into FMN, and removal of internal quenching of fluorescence by the AMP moiety.
Use of absorption spectroscopy to monitor ligand binding, redox reactions at equilibrium
The flavin absorbance spectrum is sensitive to: Redox state Ionization state of isoalloxazine positions, which is in turn sensitive to environment (protein, ligands, ...) Flavin ox (Yellow) Flavin hydroquinone (leuco) R R H O O N N CH 3 CH 3 N N NH NH CH 3 N CH 3 N O H O R R O N N CH 3 O N N CH 3 + NH NH CH 3 N CH 3 N O - O - H R R - O O CH 3 N N CH 3 N N NH NH CH 3 N CH 3 N O O H Neutral semiquinone (Blue) Anionic semiquinone (Red)
Anaerobic NADPH Titration of MICAL-MO NADPH NADP + E ox E ox •NADPH E red •NADP + E red 0,20 12 A 340 0,15 8 Y*E Absorbance A 457 4 0,10 0 0 10 20 30 40 0,05 NADPH, uM 0,00 300 400 500 600 700 800 Wavelength nm • FAD hydroquinone is formed without formation of intermediates • K eq = 0.591*10 6 ΔG ° ’= -32 kJ/mol ΔE ° ’ = 0.166 V • E m of the FAD/FAD hq couple: -0.150 V
Use of absorption and fluorescence spectroscopies to monitor enzyme-catalysed reactions under steady-state and pre-steady- state (rapid reaction) conditions.
Kinetic measurements aim to define the mechanism of a reaction or process and its free energy profile. k 1 k 2 k 3 k 4 k 5 k 6 k 1 k 3 k 5 E + S ES EP E+P k 2 k 4 k 6
Initial velocity measurements under steady-state conditions allow to determine the kinetic parameters V and K M for the substrates, which depend on the rate constants that govern the individual reaction steps. Velocity measurements under pre-steady-state conditions allow to determine directly the values of the rate constants that govern the individual reaction steps.
Initial velocity measurements of the enzyme-catalyzed reaction under steady-state conditions are carried out, under a variety of conditions, - to quantify the enzyme and - to obtain information on the enzyme function, the reaction mechanism, regulatory mechanisms, the active enzyme form. Substrates Products d[S] d[P] v = - = dt dt
The Michaelis-Menten Equation relates the initial reaction velocity to the concentration of (active) E forms, [S] and «groups» of rate constants of elementary reaction steps The Assumptions of the steady-state model - [E] << [S o ] - Measure v o (initial velocity) when [P] = 0 - v = k 3 *[ES] - [ES] = constant k 1 k 3 E + S ES E+P k 2 V max = k 3 [E t ] V max [S] v o = k 2 + k 3 K m + [S] K m = k 1 k cat = V/[E t ] = turnover number V/K = catalytic efficiency
Evaluation of k cat and and k cat /K values may help establishing the physiological reaction of novel enzymes Tawfik, D., et al. Proc Natl Acad Sci U S A. 2014 May 20; 111(20): E2078–E2079. Published online 2014 Apr 23. doi:10.1073/pnas.1401685111
Robust assays of enzyme activity are needed to gain information on: - The enzyme substrates/products - Inhibitors testing - Definition of the enzyme mechanism (also for drug design) - Screen and analyse engineered forms (also for biotechnological applications) Information on the enzyme are gained by correlating changes of the steady-state kinetic parameters V (or kcat) and Km as a function of (e.g.): - substrate(s), their concentration, - pH, ions, solvent viscosity, - effectors (inhibitors/activators), - temperature - isotopic substitution of defined positions of substrates (substrate kinetic isotope effects) and solvent (solvent kinetic isotope effects).
d[S] d[P] Products Substrates v = - = dt dt Continuous spectrophotometric assays are very handy: no [P] sample manipulation, direct observations, often high sensitivity, reproducibility. Discontinuous methods for the [P] Detection and Quantitation of the reagents are time-consuming, requiring: ( Chromatographic) t separation of the reaction components at different times followed by Detection and Quantitation of the reaction components Time (min) by UV, Vis Absorbance, Fluorescence; Conductivity; Radioactivity, ….
Example: Monitor NAD(P)H oxidation (or NAD(P) reduction) in reactions catalyzed by dehydrogenases/ reductases, oxidases, (mono)oxygenases MICAL NADPH + H + + O 2 NADP + + H 2 O 2 Monitoring the entire spectrum can help troubleshoot: aggregation , precipitation of substrates/products; artifacts Calculate initial velocity from absorbance changes at 340 nm with known extiction coefficient of NAD(P)H
Coupling the reaction of interest with an indicator reaction with substrates/products suitable for a spectrophotometric assay is very handy. For consecutive reactions: A →B → C If v B → C >> v A →B , then v A → C = v A →B Depending on the products several indicator rxns can be used.
H 2 O 2 production is often measured by coupling it to Horseradish Peroxidase in the presence of Amplex red by fluorescence, but possible artifacts may arise from the specific reaction. H 2 O 2 H 2 O Resorufin Amplex red
Possible artifacts: The HRP coupled assay of MICAL-MO NADPH H 2 O 2 Amplex Red MICAL HRP NADP + O 2 Amplex Red ox H 2 O NADPH oxn Reconstruct assay from figure: 220 12 8.0 s -1 215 10 210 8 [NADPH], µM [H 2 O 2 ], µM 2.9 s -1 205 6 200 4 10.4 s -1 195 2 15.6 s -1 190 0 0 2 4 6 Amplex red oxn Time (min) Turnover number: 15.6 or 10.4/s by monitoring NADPH oxidation vs 8 or 2.9/s by monitoring Amplex red oxidation Figure 5, PNAS, 2005
Substrate H 2 O 2 Dye Enzyme HRP O 2 Substrate ox Dye ox H 2 O Controls: MICAL‐MO/o‐dianisidine - HRP, Amplex red and H 2 O 2 enhance NADPH oxidation - NADPH inhibits HRP GO/Glc/o‐dianisidine - NADPH lowers the amount of H 2 O 2 detected at the end of the reaction Conclusions: - the spectrophotometric coupled assay cannot be used to assay MICAL NADPH oxidase activity - Rather just measure NADPH oxidation at 340 nm
MICAL controls axon growth in response to semaphorins binding to their Plexin receptor Inhibition of MICAL may promote nerve regeneration after spinal chord injury EGCG, as a specific inhibitor of MICAL1 could be used a sa drug to promote axon regeneration (-) epigallocatechin gallate EGCG mimics MICAL LOF mutants by acting as a specific inhibitor of MICAL-MO function EGCG is a specific and potent noncompetitive inhibitor of mMICAL- MO with Ki, 0.5 mM:
Effect (and structure) of (-) epigallocatechin gallate (EGCG) is very similar to the effect of xanthofulvin , a potent inhibitor of Sema3A, which has been shown to promote recovery form spinal cord injury in rats SM-216289 EGCG
The activity assay is critical to gather sound data ECGC as a catecol EGCG causes MICAL scavenges H 2 O 2 denaturation as revealed by enzyme titration 0.20 MICALHis start 2.5 uM EGCG 0.15 5 uM EGCG Absorbance 12.5 uM EGCG 25 uM EGCG 0.10 50 uM EGCG 75 uM EGCG 150 uM EGCG 0.05 275 uM EGCG 0.00 300 400 500 600 700 800 wavelength, nm Quantitation of H 2 O 2 (50 μM) with HRP/o- dianisidine in the presence of EGCG
By monitoring NADPH oxidation at 340 nm (no HRP, no dye but with hMICAL), EGCG is a much less potent inhibitor than previously reported 8 4 6 3 v/E, s-1 v/E, s-1 4 2 2 1 0 0 -0.02 0 0.02 0.04 0 20 40 60 80 100 [NADPH], µM 1/[NADPH], 1/µM - NonCompetitive inhibition but K is = K ii = 17 µM >> 0.5 µM - Excess inhibition at high NADPH due to enzyme denaturation?
Assay set-up requires optimization of - Temperature - Buffer composition (type of ions, ionic strength, viscosity) - pH - Added ligands, ions, coenzyme/cofactors If there is a choice between absorbance- and fluorescence-based assay, select the assay method
MICAL NADPH oxidase reaction is sensitive to ionic strength and the type of anions. Strong effect on V/K NADPH mainly due to effect on Km due to: Competition between anions and NADPH Electrostatic effects 500 100 2.0 - Log(kcat/KNADPH) KNADPH/kcat µM*s KNADPH/kcat µM*s 400 80 1.5 300 60 1.0 200 40 0.5 100 20 0 0.0 0 0 100 200 300 0 2 4 6 0 20 40 60 80 100 I1/2, mM1/2 I, mM I, mM Buffer; Buffer: imidazol-chloride ( ), Buffer :20 mM Hepes/NaOH, pH 7.0: Hepes/NaOH buffer ( ), ●, Na acetate; , Mg acetate; imidazol-acetate ( ), Tris-chloride ( ) , NaCl; , KCl; , KCl 2 ; Bis-Tris-acetate (●). Tris-acetate ( ). , sodium phosphate. Fit to Debye-Huckel equation Design mixed buffer for pH studies to minimize ions and I effects, keep I under control in expts.
pH dependence of steady-state kinetic parameters of the NADPH oxidase reaction of MICAL forms need to be studies in a mixed buffer that guarantees a constant ionic strength
Most enzyme reactions are well described by the Michaelis-Menten equation V max [S] v o = K m + [S] k 2 + k 3 V max = k 3 [E t ] K m = k 1 k cat = V max /[E t ] = turnover number V max / K m or k cat / K m = catalytic efficiency
Deviations from the Michaelis-Menten equation are informative Two enzyme forms? 4000 3500 3000 2500 Substrate inhibition? 2000 v 1500 1000 Allosteric activation 500 0 0 50 100 150 200 250 [S]
Dependence of v from [E T ]: deviations from the predicted linearity are informative Expected and Most common behavior Non- v Reaction too fast enzymatic to measure the reaction initial velocity; Monomer/dimer equilibrium; dimer is inactive A tight binding inhibitor in the reaction mixture, Monomer/dimer equilibrium and [E T ] the monomer is inactive
Use activity assays of HIV protease to determine the dissociation constant of the (active) dimer + 2 M D (dimer, active)
Selection of the activity assay
A fluorescent substrate to measure HIV1 protease activity C C N + HIV1 protease N N Ser-Nle-Ala-Glu-pNitro-Phe-Leu-Val-Arg-Ala-Lys-His-Abz Quenching of Abz fluorescence by nitroTyr
The alternative absorption-based assay for HIV1 protease exploits the effect of changes of nitroTyr environment during the reaction. The observed absorbance changes are smaller than fluorescence C changes C N + HIV1 protease N N Ser-Nle-Ala-Glu-pNitro-Phe-Leu-Val-Arg-Ala-Lys-His Substrate Spectra: before and after incubation with HIV‐1 protease HIV 1Protease Activity 0.800 0.34 0.700 ε≈ 0.50 mM -1 cm -1 0.32 0.600 Absorbance, 310 nm 0.500 0.3 0.400 Abs (A.U.) 0.28 0.300 0.26 0.200 0.100 0.24 0.000 0.22 200 250 300 350 400 450 500 ‐0.100 0.2 ‐0.200 Wavelenght (nm) 0 200 400 600 800 1000 1200 1400 1600 1800 time (sec) Substrate Substrate + HIV‐1 Protease Spectra Difference
Absorption-based assay allows to explore a broader substrate range for V and Km determinations Absorbance-based assay Fluorescence-based assay Apparent turnover number (F in arbitrary units) Actual turnover number (ε is known) Buffer :100 mM Na Acetate, pH 5.00, 1mM EDTA, 1 mM DTT, 100 mM NaCl
Activity assays allow to monitor the dissociation of the HIV-1 protease dimer. + 2 M D (dimer, active) [M] 2 v = k cat * [Dimer] K d = [E tot ] = 2*[D] + [M] [D] 1.8 1.6 1.4 v/[Et], s -1 100 mM Na Acetate buffer, pH 5.0, 1 mM Buffer STD Activity (U/ml) 1.2 pH 5 EDTA, 1 mM DTT, 100 mM NaCl U/mL 1 Buffer 0.8 Bachem pH 10 mM Na phosphate buffer, pH 6.5, 1 mM 6.5 0.6 EDTA, 10% glycerol, 75 mM NaCl; 0.4 calculated Kd, 215 nM 0.2 0 100 200 300 400 500 600 [E] nM
Steady-state kinetic studies to monitor binding of activating proteins
Flavin ox Flavin red R R N O N N O N NAD(P)H N N N N O O H SOH + H 2 O O 2 S (monooxygenaseactivity) R O N N H 2 O 2 Flavin ox + N (oxidaseactivity) N O O H OH Flavin ox + O 2.‐ 4a‐hydroperoxy‐Flavin
Rab proteins are physiological modulators of MICAL activity by binding to its C-terminal region Rab8.GppNHp Kd, 55 nM Kd, 480 nM MICAL1 C‐term / Rab35, 1:1 complex; Kd 6‐13 µM
Effect of Rab8.GDP and (active) Rab8.GppNHp on the NADPH oxidase activity of MICAL1 (no effect on truncated forms: the RBD is in the MICAL1 C-terminal region) 0.8 0.24 v/E, s-1, no Rab8.GppNHp 0.6 0.18 v/E, s-1 0.4 0.12 k cat -Rab , 0.35 s -1 0.2 k cat +Rab , 1.1 s -1 0.06 K Rab , 9 µM K Rab , 92 µM k M,NADPH , 1.1 mM 0.0 0.00 0 400 800 1200 1600 [NADPH], µM No coelution; apparent mass of MICAL1 between that of monomer and dimer
Analysis of the aggregation state of MICAL and MOCHLIM and of complex formation with Rab8.GppNHp Porod volume/1.6 62.5 90.6 132.5 146 55 kDa 68.2 kDa 86.3 kDa 112 kDa
SAXS-based modelling of MICAL1 and its complexes with Rab8.GppNHp: MICAL1 is a monomer; it forms a 1:1 complex with Rab8.GppNHp. MOCH MOCHLIM MICAL1 MICAL1+Rab
Rapid reaction kinetics
Initial velocity measurements under steady-state conditions allow to determine the kinetic parameters V and K M for the substrates, which depend on the rate constants that govern the individual reaction steps. Velocity measurements under pre-steady-state conditions allow to determine directly the values of the rate constants that govern the individual reaction steps
Rapid reaction kinetics allow to directly measure rate constants to study: • Chemical reactions • Folding/Unfolding • Protein-protein, Protein-ligand interactions • Conformational changes A multistep process
To measure individual reaction steps we need to rapidly monitor changes in a signal that is related to the identity and concentration of each species that may be formed during the process (which may be fast). k 2 k 1 C A B A = A o e -k1t I ,A = [A]* A B = A o k 1 /(k 2 -k 1 )(e -k1t - e -k2t ) I B = [B]* B C = A o [1 + 1/(k 1 - k 2 )(k 2 e -k1t - k 1 e -k2t ) I C = [C]* C Isosbestic points
Requirements of rapid kinetics [Enzyme] : µM, mM vs nM, µM for steady-state [Substrate] : µM, mM ( [S]> 10x [E] for pseudo-first order conditions) Measuring Times: msec-sec vs sec-min for steady-state Thus, need: - large amounts of enzyme/protein & substrate/ligand - highly concentrated protein/ligand solutions - rapid mixing device - rapid measuring times (in continuous methods) - rapid data acquisition - software (expertize) for data analysis
Detection Continuous methods: Absorbance, Fluorescence, Circular Dichroism, Fluorescence anisotropy, conductivity, X-ray scattering (!), ….. Discontinuous methods (coupled to continuous flow set-up) EPR (freeze-quench) Mossbauer (freeze-quench) HPLC separation of reaction components and chemical analysis (chemical quench)
Continuous flow set-up for rapid reaction studies Tubing of different length will lead to quenching of sample at different reaction times: 1 cm = 1 msec; 2 cm = 2 msec, etc. for 10 m/s flow rate Push, 3 atm Cold isopentane Analysis: one «shot» / 1 datapoint
Stopped-flow set-up for rapid reaction studies Detector Drive Stop Mixer Push, 3 atm syringes syringe Trigger Detector Turbulent flow to ensure constant velocity across tubings High flow rate ( e.g.:10 m/s = 1 cm/msec)
Different instrument set-ups for stopped-flow Single Mixing Double Mixing Stopped-flow Stopped-flow E + S 1 ES 1 E + S Aging time, varies ES 1 + S 2 (or I, or ....) (1:1 or variable volume mixing)
The upper limit of measured rates is set by: Dead-time, Time-constant (Time Resolution), Sensitivity of detector Dead-time Time-constant (Resolution) td 2 msec td 2 msec 1.0 1.0 0.8 0.8 Absorbance Absorbance 0.6 0.6 1000/s 50/s 0.4 0.4 50/s 0.2 0.2 100/s 500/s 250/s 1000/s500/s 2000/s 0.0 0.0 0.000 0.020 0.040 0.000 0.020 Time (sec) Time (sec) Detector Push, 3 atm Detector
Flavin ox Flavin red R R N O N N O N NAD(P)H N N N N O O H SOH + H 2 O O 2 S (monooxygenaseactivity) R O N N H 2 O 2 Flavin ox + N (oxidaseactivity) N O O H OH Flavin ox + O 2.‐ 4a‐hydroperoxy‐Flavin
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