Stability of biomolecules - methods Josef Houser Autumn 2023 S1004 Methods for structural characterization of biomolecules Stability Resistance in environment Capability to retain native structure (stay folded) Capability to retain activity no structure no activity structure ? activity Stability Quick reminder-structure hierarchy Protein DNA Primary Sequence (aminoacids, N-term - C-term) Sequence (nucleotides, 5X- 3xend) Secondary a-helix, (3-sheet, turns, loops (rotation along torsion angels ^ and CD) Watson-Crick base pairing (A-T, C-G) Tertiary 3D organization of secondary motives A-form, B-form, Z-form Quartern a ry oligomerization nucleosomes Stability Quick reminder - structure hierarchy Protein DNA Primary Secondary Tertiary Sequence (aminoacids, N-term - C-term) Sequence (nucleotides, 5X- 3xend) Upon unfolding (= denaturation) these structures are lost. The primary structure remains Quarternary \7 The covalent bonds are not broken, only the non-covalent Thermodynamic consideration K U M [N] unfolded [U] Ku«l Ku »1 native state is favourable unfolded state is favourable Thermodynamic consideration M u [n] k„ = native [N] unfolded [U] K u e-AG/RT Native state is favourable when Ku < 1 -AG/RT is negative AG... Gibbs free energy T... temperature (in Kelvins) R... gas constant (8.314 J-K^-mol1) e... Euler number (2.718....) K,, « 1 native state is favourable Ku » 1 unfolded state is favourable ex 0 1 2 x (-AG/RT) Thermodynamic consideration k = M u [n] Ku « 1 native state is favourable Ku » 1 unfolded state is favourable native [N] unfolded [U] K u Temperature [K] -> always positive Gas constant = 8.314 Gibbs free energy AG... Gibbs free energy T... temperature (in Kelvins) R... gas constant (8.314 J-K^-mol1) e... Euler number (2.718....) AG/RT is negative when AG > 0 Thermodynamic consideration AG = Gibbs free energy 'U AG - Gy - GN native [N] unfolded [U] AG = AH - TAS The reaction with -AG will hapen spontaneously The state with lower G is prefered For the protein to be stable, AG of unfolding needs to be positive (in such a case GN < Gy) G... Gibbs free energy T... temperature (in Kelvins) H... enthalpy S... entropy -AG spontaneous direction is downhill proceeds unaided +AG non-spontaneous direction is uphill proceeds only if work is done Thermodynamic consideration 'u native [N] unfolded [U] AG = AH-TAS AG = GU_GN H = Enthalpy Changes in heat Energy content of the bonds broken and created hydrogen bonds, van der Waals, salt bridges, S-S AH is negative when bonds are formed S = Entropy Changes in disorder Degree of freedom of molecular movement Brown's motion movement = 1^ S AS is negative when bonds are formed Thermodynamics of unfolding N 'U native [N] unfolded [U] H = Enthalpy S = Entropy +as - unfolded state is more flexible AG = AH-TAS AG = GU"GN +ah - a lot of non-covalent interactions in folded state -as from hydrophobic effect - upon exposure of hydrophobic side chains, the water surrounding the protein forms an ordered cluster (Jcebergs") Thermodynamics of unfolding N 'U native [N] unfolded [U] H = Enthalpy S = Entropy Proteins are just stable AG = AH -TAS AG = GU_GN AG of unfolding is typically < 100 kJ mol1 (compared to energy of C-C bond 300 kJ mol1) Small changes in protein environment can significantly influence the stability Denaturing conditions H2N IMH2 o Chemicals ^ • Urea (around 8M) H2N^ ^NH2 nh • Guanidium chloride (around 6M) _____Jl^ • High salts concentration • SDS o 11 CH3(CH2)110-S-ONa 0 Extreme pH • Proteins are stable the most near their isoelectric point (pi) Temperature as denaturant Tm = melting temperature • Temperature at which 50% of the sample is unfolded • The most reliable indicator of thermal stability Raw data 20 30 40 50 60 70 Temperature [°C] 80 90 First derivative Temperature [ Temperature as denaturant Tm = melting temperature Influenced by: • Enviroment (buffer, pH, salts): in different condition, AG of unfolding is different • Presence of ligand: protein-ligand complex is more stable than protein itself Heating rate of experiment: slower heating -> lower T, standard is 1 °C/min m AG = AH - TAS Methods • Differential scanning calorimetry (DSC) • Differential scanning fluorimetry (DSF) - Thermal shift assay (TSA) • Nano-differential scanning fluorimetry (nanoDSF) • Circular dichroism (CD) DSC = Diferential Scanning Calorimetry Measures the energy absorbed or released by a sample as it is heated or cooled Gold standard for Tm determination Directly measures the thermodynamic of unfolding DSC = Diferential Scanning Calorimetry Sample cell (sample) and reference cell (buffer) are heated/cooled down at the same rate Sample absorbs/release part of the energy causing a temperature difference between the sample and reference cell DSC machine measures the energy needed to equalize the temperature 0) o CD >C = Diferential Scanning Calorimetry 40 50 60 70 80 90 Temperature (°C) 100 m the peak of the transition ac p change of heat capacity folded-unfolded sample Difference between two baselines ah cal Area of the peak (integration) ahvh The slope of the peak DSC = Diferential Scanning Calorimetry AH can be determined in two ways: • Directly by calorimetric measurement - area under the peak - AHca| • Indirectly by measureing the temperature dependance of the eqilibrium constant - vanvt Hoff method, the slope of the peak, AHvH If the difference between AHca)and AHvH is observed, it indicates that the reaction is more complicated - presence of intermediate state DSC machines VP-DSC 1 sample at a time 0.8 ml sample at 0.1-2 mg/ml Identical buffer in reference cell necessary (dialyses, lyophylization) Degasing required Auto PEAQ-DSC Automated version (up to 282 samples in a row) 0.2 ml sample at 0.1-2 mg/ml Identical buffer in reference cell necessary (dialyses, lyophylization) DSC = Diferential Scanning Calorimetry Pros: Cons: Direct measurement of • Time consuming thermodynamics . High sample consumption Label free Gold standard forTm measurements Suitable for proteins, nuclei acids, lipids, polymers... Fluorescence It is a physical phenomenon in which "light" is emitted by a substance that has previously absorbed electromagnetic radiation excitation light fluorophore emitted light gamma rays x-rays ultraviolet ■ rays I infrared rays radar FM TV shortwave AM 1 II 10" 10" 10.io 10* 10s 10" 10s 1 10-' 10- Visible Light wavelength (meters) Nanometers (nm) TSA = Thermal Shift Assay Also known as differential scanning fluorimetry ( = DSF) High-throughput (96 well plates) No specialized machine - uses termocycler for RT-PCR Measures changes of fluorescence of the sample in temperature gradient • Commercial dyes • GFP-tag TSA = Thermal Shift Assay Commercial dyes (e.g. SYPRO Orange, bis-ANS, Nile Red): Provide fluorescent signal only when they interact with hydrophobic residues of the unfolded protein Limitations: • Target protein do not have significant hydrophobic patches on the surface • The target protein is folded at the begginning of experiment • Dye do not bind to target protein • Dye do not react with experimental buffer TSA = Thermal Shift Assay Fully denatured protein a (maximum dye binding) TSA = Thermal Shift Assay GFP-tag: GFP signal changes with its close environment, reports the unfolding of target protein Limitations: - • Tm of GFP is around 75 °C - only usable for less stable proteins • Potential changes in conformation or oligomeric state of target protein after adding a GFP-tag TSA in practice Protein and dye incubated in 96 well plates Changes in fluorescence monitored 1E6 -i (a.u.) 8E5 ■ CD 6E5 ■ O c CD O 4E5 ■ C/5 CD O ZJ 2E5 ■ U_ 0 ■ 30 40 50 60 70 80 90 Temperature (°C) B 1E6 ■ (a.u.) 8E5 ■ 0 6E5 ■ o c CD Ü 4E5 ■ w CD o ZJ 2E5 ■ u_ 0 ■ r « m 30 40 50 60 70 80 Temperature (°C) 90 30 40 50 60 70 80 Temperature (°C) doi:10.1002/0471140864.ps2809s79 TSA = Thermal Shift Assay Pros: Cons: • Quick • Needs dye • High-throughput • Usage of GFP-tag lim • Excelent for sample comparison • Data analysis • Affordable instrumentation Intrinsic fluorescence of proteins fluorophore Tryptophan (Trp, W) Tyrosine (Tyr, Y) Phenylalanine (Phe, F) NH2 OH Aromatic amino acids Intrinsic fluorescence of proteins fluorophore Tryptophan (Trp, W) Ty "OH Aromatic amino acids Intrinsic fluorescence of proteins in UV region (A = 300-360 nm) nanoDSF = nano Diferential Scanning Fluorimetry Measures changes of intrinsic fluorescence of the sample in temperature gradient High-throughput (48 or 96 samples in 1 run) Low sample consumption (10 Ideal for optimal condition screening nanoDSF Intrinsic fluorescence of proteins (UV region, A = 300-360 nm) is changing according to the local environment 330 nm 350 nm "to c CD CD U c CD u 00 CD o Z5 300 320 340 360 Emission wavelength (nm) hydrophobic maximum at 330 nm hydrophilic maximum at 350 nm nanoDSF Aromatic aminoacids (W, Y, F) are hydrophobic and are typicaly located inside the folded protein o With increasing temperature the protein is unfolded o W, Y, F are exposed on the protein surface Changes in fluorescence nanoDSF in practice Protein in buffers Put into a capillary 48 samples nanoDSF in practice Design of experiment: Temperature gradient 20 - 110 °C Heating rate 1 °C/min quicker higher Tm slower lower Tm Prometheus nanoDSF in practice pH = 2 Tm = 63.3 °C pH = 3 Tm = 78.8 °C pH = 7.5 Tm = 97.1°C 20.000 30.000 40.000 50.000 60.000 70.000 80.000 90.000 100.000 110.000 nanoDSF in practice 1 Z 3 4 s 6 7 8 9 10 11 12 A 20 mMTris. 150 mM NaCl pH7,5 50 mM malaate pH2.0 100 mM glytina pH3,0 100 mM formate pH4,0 100 mM citrate pH 5,0 100 mM cacodylate pH 6.0 100 mM Hepes pH7,0 100 mM bidne pHB.O 100 mM CHE? pH 9,0 50 mM borate pH 10.0 100 mM CAPS pHll.O 100 mM f:lir^|il'.;li-pHLT.,0 56.7 97.1 63,3 78.7 93.0 99.4 95-2 96.9 90.5 86.3 75.2 64.3 B 100 mM acetate pH4.0 100 mM acetate pH 4.5 LOOmM acetate pHB.O 100 mM MES phS.5 100 mM MEi pHt.O 100 mM MES pHS.5 100 mM Na phosphate pH 7.0 100 mM K phosphate pH7.5 100 mM Iris pHfl.O 100 mMTrls pHS.5 100 mM glycine pH9.0 100 mM glycine pHQ.S 93,9 95.6 96.7 96,5 96.8 95,9 70.2 70.5 94.0 92.1 91,9 88.0 C 100 mM ME?. 100 mM Natl, pHG.O 100 mM ME5, 2wi«M N.i. 1. pH6.0 100 mM ME5, N.i. 1. pH6,0 100 mM ME?. 1000 mM NaCl, pHG.O 100 mM Na ihosphate, 100 mM NaCl, pH7,0 100 mM Na filif:1-|il 200 mM NaCl, pH7.0 100 mM Na phosphate, 500 mM NaCl, pH7,0 100 mM Na f.hi:-- pi 1000 mM NaCl, pH7,0 100 mM Iris, 100 mM NaCl, r:ll 10 100 mM Tris, 2winM N.i. 1. pHB.O 100 mM Iris, 500 mM NaCl, pHS.O 100 mM Iris, 1000 mM NaCl, pHB.O "■- 97.1 96.9 "■- *■" 93.7 93.3 93.0 93.1 94.9 D /Li r\W 1 ■ \, 150 mM NsCL 100 uM Cadi, pH 7.5 10 mM HE PES, ISO mM N-nCI, 1. ]H T.S 12 mM P-EiS. 130 mM NaCI, 2.7 mM KCI, pH7.5 12mM PBS, 0.0596 Tween 30, pH 7i 200 mM Imidazole, pH 7.5 0.05% Tween id S% glycerol 5 mMbME 5% DMSO 5% trehalose 20 mM argjnlne, 20 mM glutamlne SmM EDTA 96.1 95.2 -,- 94,7. 96.4 9B.2 96.2 97.8 97.6 95.0 95.3 nanoDSF Pros: Cons: Quick High-throughput Low sample consumption (10 \x\) Low concentration (0.1 - 1 mg/ml) No labelling Excelent for sample comparison User friendly instrumentation • Only for proteins • W (Y, F) in sequence necessary • Sensitive to capillary purity • Delicate manipulation with capillaries Applications Thermal stability determination Ligand screening Buffer optimization for purification and storage Optimization of crystallization conditions Batch to batch comparison Comparison DSC TSA nanoDSF sample proteins, nucleic acids, lipids, polymers proteins proteins Sample consumption high low low High-throughput no yes yes Automation yes no no Enthalpy yes no indirect Fluorescent dye no yes no Literature Protein unfolding: Konnermann L: Protein unfolding and denaturants doi: 10.1002/9780470015902.a00030004.pub2 DSC: Chiu M.HV Prenner E.J.: Differential scanning calorimetry: An invaluable tool for a detailed thermodynamic characterization of macromolecules and their interactions, doi: 10.4103/0975-7406.76463 nanoDSF: Alexander CG, Wanner Rv Johnson CMV et al.: Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings doi: 10.1016/j.bbapap.2014.09.016 TSA: Gao KvOerlemans Rv Groves M.R.: Theory and application of differential scanning fluorimetry in early-stage of drug discovery. doi: 10.1007/sl2551-020-00619-2. Biomolecular I nteractions and Crystallography Core Facility bic@ceitec.cz bic.ceitec.cz UNI O CF Head Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz