Methods for 2D protein structure Josef Houser Autumn 2023 S1004 Methods for structural characterization of biomolecules Secondary structure (2D) • Arrangement of primary building blocks • Protein: • α-helix, 310-helix , π-helix • β-strand / β-sheet (parallel, antiparallel) • Turns, loops • Random coil • Nucleic acid: • Helix • Hairpin (stem-loop) • Pseudoknot Secondary structure (2D) determination • Several levels of information • Presence/absence of 2D structure • Relative amount of 2D elements • Assignment of 2D structure to specific regions/amino acids • Experimental approaches: • Stability methods • Spectroscopic experiments • Analysis of 3D structure • Prediction in sillico Denaturation insight • Only folded protein can denature • Stable 3D structure is possible due to defined 2D structure • Methods: • Differential scanning fluorimetry (DSF) – Thermal shift assay (TSA) • Nano-differential scanning fluorimetry (nanoDSF) • Differential scanning calorimetry (DSC) Spectroscopic methods • Analysis of sample in solution • Averaging of signal in studied volume • Techniques: • Circular dichroism (CD) • Infrared spectroscopy (IR, FTIR) • Raman spectroscopy • Nuclear magnetic resonance (NMR) Circular dichroism spectroscopy (CD) • CD is the difference in absorption of left and right circularly polarized light https://chem.libretexts.org/ ΔA = AL − AR = Δεcl = (εL−εR)cl Circular dichroism spectroscopy (CD) • Typical maxima and minima for each secondary structure in far-UV region (180 – 250 nm) • Resulting spectrum is a linear combination • Ratio of individual 2D elements can be calculated 2 components α-helix, β-sheet 3 components α-helix, β-sheet, other Circular dichroism spectroscopy (CD) • Information content of spectrum depends on the range of wavelengths – the more, the better • Typically achievable 190 – 260 nm 5 components helixes, sheets, turns, other 6 components helixes, sheets, turns, other 7-8 components helixes, sheets, turns, other Søren Hoffmann, Aarhus Uni Circular dichroism units Quantity Equation Units Typical values Absorbance A = log10(I0/I) – 0.1 – 1.5 Molar absorption (extinction) coef. ε = A/c l L mol-1 cm-1 10 000 – 1 000 000 (per monomer) Circular dichroism ΔA = AL – AR – 10-4 – 10-3 Molar CD Δε = ΔA/c l L mol-1 cm-1 Elipticity θ = 32 980 ΔA mdeg 1 – 100 Molar elipticity [θ] = ΔA/(10 c l) deg cm2 dmol-1 Mean residue ellipticity (MRE) [θ]MRW = ΔA/(10 cAA l) deg cm2 dmol-1 I0 – intensity of incoming light I – intensity of outgoing light c – molar concentration [mol L-1] l – cuvette length [cm] Data evaluation • Based on purpose • Basic processing in SpectraAnalysis SW • baseline subtraction • unit conversion • similarity comparison • Secondary structure content – external SW needed Secondary structure content analysis Various programs/tools for data analysis Various analysis methods (Selcon, Contin, CDSSTR) Based on analysis of highly reliable CD spectra of proteins with known 3D structure Accessible online for free (ev. registration needed) • Dichroweb – http://dichroweb.cryst.bbk.ac.uk/ • BeStSel – http://bestsel.elte.hu/ • K2D3 – http://cbdm-01.zdv.uni-mainz.de/ 11 PCDDB 12 http://pcddb.cryst.bbk.ac.uk/home.php • CD spectra database (Protein Circular Dichroism Data Bank) • Alows for spectra view, download • Linked to other resources Instrumentation 13 • Table-top instruments • Jasco • Applied Photophysics • Synchrotron CD • Accessories • Temperature control • Fluorescence measurement • Stop-flow Chirascan (Applied Photophysics) J-815 (Jasco) J-1500 (Jasco) CD beamline (Aarhus, Denmark) Sample 14 • Purity • All protein components contribute to signal • Non-protein chiral contaminants • Concentration • CD signal magnitude is affected by concentration • Change of concentration with time (? cuvette sealing) • Buffer • Measuring at short wavelengths – buffer absorption Buffer compatibility Buffer / solvent Lower wavelength cutoff for 1 mm cell dD2O 175 dH2O 180 10mM Na-phosphate 182 50mM NaF <185 150mM NaClO4 <185 10mM K-phosphate, 100mM KF 185 100mM Na-phosphate 190 150mM (NH4)2SO4 190 100mM NaCl 195 50mM Na-borate 195 Ethanol (100%) 195 PBS 200 100mM Tris-HCl 200 100mM MES 205 50mM Na-acetate 205 4M guanidine-HCl 210 4M urea 210 100mM PIPES 215 100mM ammonium citrate 220 150mM NaNO3 245 DMSO (100%) 252  ✓ ✓✓ Søren Hoffmann, Aarhus Uni Concentration determination 16 • Error in concentration reflects in CD accuracy • Absorbance A280 • Require accurate (!) measurement using correct ε • Relies on Trp/Tyr content • A205 • ε205 >> ε280 (HEWL lysozyme: MW 14.3 kDa, ε280 = 37 500, ε205 = 558 300) • Less dependent on Trp/Tyr content • Is measured directly during CD experiment CD cuvettes • Rectangular quartz cuvettes • Single piece: 0.5 mm, 1 mm, 2 mm, 5 mm, 10 mm • Sandwich: 0.01mm, 0.1 mm, 0.2 mm • Special: 5 mm fluorescence, 10 mm low volume, • Cylindrical quartz cuvettes • Single piece: 0.1 mm, 0.2 mm, 0.5 mm, 1 mm, 2 mm, 5 mm, 10 mm • Sandwich: 0.1 mm, 0.2 mm • Special cuvettes • Ultra-low volume nanodisc • Glass cuvettes for non-protein applications Specials – CD melting • Changes in 2D structure with temperature • Temperature interval scan • Full spectrum in defined temperature points • Slower • Full understanding of changes in structure • 3D plot available • Variable temperature measurement • Fixed wavelength • Faster, suitable for melting curve fitting Intensive cuvette cleaning necessary !!! J Seelig & H-J Schonfeld 2016 Infrared spectroscopy (IR) • Absorption spectroscopy of infrared light coupled with Fourier transformation (FTIR) • Characteristic protein/polypeptide absorption bands Kong J and Yu S, 2007, Acta Biochim Biophys Sin Amide I region • Amide I region (1600-1700 cm-1) • Mainly caused by peptide C=O vibration • Highly sensitive to secondary structure • Water absorption at 1600 cm-1 • 10x stronger than protein signal • Measurement in D2O • Thin layer (<10 μm) – high protein conc. • Precise reference subtraction needed • ATR-FTIR – attenuated total reflectance – 1-2 μm penetration Amide I region • Peak overlap for individual 2D structures • Multiple bands • “Increase” of resolution needed • Several methods, e.g. second derivative analysis Kong J and Yu S, 2007, Acta Biochim Biophys Sin FTIR instrumentation • IR spectrometers – various producers and variants • Quartz cuvettes not IR transparent • Other materials used: CaF2, ZnSe Confocheck (Optik Instruments) IRAffinity (Shimadzu) Microfluidic modulation spectroscopy (MMS) • IR spectroscopy with microfluidics for sample delivery • Broader concentration range than FTIR, < 0.5 - > 100 mg/ml • Suitable for pharmacology, e.g. antibody characterization • Promissing for future development AQS3 Pro (RedShiftBio) Comparison CD IR Wavelength UV (180-250 nm – proteins, 250-350 nm – NA) IR (5900-6250 nm = 1700-1600 cm-1) Sample concentration 0.1 – 5 mg/ml 0.5 – 200 mg/ml Sample volume 5-200 ul 300-1500 ul Advantages Well developed Low sample consumption Broad concentration range Light scattering insensitive Disadvantages Buffer interference Sample consumption Data transformation Raman spectroscopy • Molecular vibrations measured by inelastic scattering in IR • Raman spectroscopy and Raman optical activity • Typically high protein concentration (> 10 mg/ml) J Peters et al 2016 Nuclear magnetic resonance (NMR) • Spectra of structured proteins are more resolved • Chemical shift correlates to secondary structure arrangement • Peak integration (1D-COSY) or counting (2D-COSY) C Lundin 2011 Mielke & Krishnan 2009 2D structure from 3D structure • Most precise • Calculated from back bone torsion angles • Low-resolution structures – estimation based on typical shapes in electron density Wang et al 2016, eLife 2D structure application • Protein characterization • Batch-to-batch quality check • Analysis of protein folding • Measurement of protein interaction • Protein stability assessment with respect to environment variables – temperature, pH, ionic strength • Protein identification (based on PCDDB database spectra) • Protein classification within SCOP database (Structural Classification of Proteins) • Assistance in 3D structure determination 28 Further reading • Norma J. Greenfield: Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006; 1(6): 2876–2890. doi: 10.1038/nprot.2006.202 • https://www.niu.edu/chembio/_pdf/analytical-lab/cd/handout.pdf • Jilie Kong and Shaoning Yu: Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures. Acta Biochim Biophys Sin 2007; 39(8): 549-559. doi: 10.1111/j.1745-7270.2007.00320.x • Andreas Barth: Infrared spectroscopy of proteins. Biochim Biophys Acta 2007; 1767(9): 1073-1101. doi: 10.1016/j.bbabio.2007.06.004 • Steven P. Mielke and V. V. Krishnan: Characterization of protein secondary structure from NMR chemical shifts. Prog Nucl Magn Reason Spectrosc 2009; 54(3-4): 141-165. doi: 10.1016/j.pnmrs.2008.06.002 29 Questions? 30 ? CF Head Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz bic@ceitec.cz bic.ceitec.cz Biomolecular I nteractions and Crystallography Core Facility