Sample in structural biology Josef Houser Autumn 2023 S1004 Methods for structural characterization of biomolecules Biomacromolecules Biomolecules are natural parts of living organisms. Macromolecules typically compose of thousands to millions of atoms. Small molecules compose of hundreds of atoms or less. Molecules are essential parts of matter. They consist of atoms that are linked through covalent bonds. Biomolecules ^ Biomacromolecules Macromolecules Basic chemical composition of biomacromolecules Heteropolymers consisting of various subunits Macromolecule Building blocks Type of bond Scheme Protein Amino acids Peptidic OR 0 ' ^NH Y ^NH 0 R Nucleic acid Nukleotides Esteric 0 °H 0// OH Polysaccharide Monosaccharides Glycosidic OH OH Amino acids X ,OH H£N jl^ H2N Glycine O Phenylalanine O QH Me H H'N if Alanine 0 H2N Mettaonine O Valine O H2N Leucine O H2N Isoleucine O Tryptophan O Serine O H2N Threonine O H2N Asparagine O OH "N" H Proline y W Lysine O H2N Histidine O Glycine Alanine Valine Leucin Isoleucine Aspartic acid Asparagine Glutamic acid Glutamine Arginine Lysine Histidine Phenylalanine Serine Threonine Tyrosine Tryptophan Methionine Cysteine Proline Selenocysteine Pyrolysine Gly Ala Val Leu lie Asp Asn Glu Gin Arg Lys His Phe Ser Thr Tyr Trp Met Cys Pro Sec Pyr G A V L 1 D N E Q R K H F S T Y W M C P U 0 B X-Any Types of amino acids Amino acids with similar properties may substitute each other in protein Isoleucine Leucine Nucleic bases NH: O N V NH (Li NH^O NH^N^V Adenine Cytosine o H3C NH NH Thymine N NH2 Guanine o " NH NH Uracil adenine cytosine guanine thymine uracil A C G T U Nucleic base Adenine < N Nucleoside H0_, Adenosine Nucleotide Adenosinmonophosphate AMP 0 II HO—P—O- 1 OH { N OH OH N ❖ AltN Qu IN Ac Rha/JAc DeoxyHexNAc /k Kha fthaNAc 2k 3 3 AltA AHA AHM TaIN SdAlt TalA o IduN IdoA DIDeoxyHexose OH Tyv Abe Par Dig Col ; i Pentose Nonulosonate ♦ Lvx & Kyi ★ Rib ★ Neu5Gc O Neu ♦ Assigned (1} Bat LDManHep Kdo CJ □ha DDManHep MurNAc MurNGc O Muf Assigned (2} Api * Fru Tag o Sor _ Psi * Fur Combinatorics Structural variability reflects length, variability of units and variability of bonds Polymer Protein Nucleic acid Polysaccharide Number of various basic units 20 (22) 4 (DNA) 4 (RNA) 150 (identified) Number of possible bond types 1 1 2x4 (for hexose) Theoretical number of possible molecules consisting of 2 units 22 x 22 = 484 150 x 150 x 8 = 180 000 Structural hierarchy ID ADSQTSSNRAGEFSIPPNTDFRAIFFANAAE QQHIKLFIGDSQEPAAYHKLTTRDGPREATL NSGNGKIRFEVSVNGKPSATDARLAPINGK KSDGSPFTVNFGIVVSEDGHDSDYNDGIVV LQWPIG primary (sequence) quaternary CCC CCEEC CCCCC C C C C C C EEEE C C C C C EEEEEEECCC CC DS Q EPAAr HKLTTRDC FP. EA 7LN ECNCKIPFEV E V?iCK FS ■■<1 70 80 CCH HEEEECC-CCC CCCCC CEBLEEBLBLEBLCCKCCCCCCCee ATOAPLAFINGKKS CGSFFTWFGIWS eOGHOSOVNOG I SO 100 110 secondary tertiary Sample's life Storage Expression system Media formulation Modifications Procedures Co-purification Temperature Concentration Labelling Immobilization Toxicity Biodegradation Forms of sample - solution, powder, crystal, surface-bound 10 Quality of sample • All properties that relates to sample state and determining its behavior 11 Sample requirements • Minimal requirements: • Conditions that sample has to meet in order to give some results • Differ heavily for individual techniques • Minimal requirements for specific technique do not ensure good sample mi Example Minimal requirements: 0.5 mg/ml sample 450 ul volume No DTTin buffer 12 Concentration Mass concentration (p;, y): [mg ml"1] = [ug ul_1] Molar concentration (c): [M] = [mol I1], [mM], [uM] Conversion: M, Molar mass - inaccurate knowledge cause errors! 13 Concentration determination Method Nitrogen content (e.g. Kjeldahl) UV absorbance at 280 nm Bradford (Coomassie Brilliant Blue) Bicinchoninic acid UV absorbance at 205 nm Absolute (golden standard) Fast, easy, low sample consumption, no calibration Easy, fast Less buffer dependent Less sequence dependent, + the same as A- Time, sample and equipment demanding Sequence dependent, buffer influence, (inaccuracy in I, e) Standard dependent (calibration), sequence dependent, buffer influence Standard dependent (calibration), more time demanding Buffer absorbance 4280 Ideal sample properties Defined (chemically, biologically, conformationally) Pure (contamination by small molecules, macromolecules) Homogeneous (micro-/rriacro- heterogeneity) Stable (storage, time-demanding analysis) Sample identity • Exact composition of sample (sequence, modifications, cleavage) • Influence on MW, pi, interactions Covalent oligomerization Glycosylation Signal peptide )( iaHIA99^DAISNSN9AA^DSHAAIISiaN9ANAIANANSAiaSSS¥9AaSJ iswiAAasaATsaaiiiaaiiNNDANsDaasaAassaaisaisaAYSwaYDSj ¥I¥iaDASDlA9aiAAI¥NINa9¥aAlDaWI9SIH¥S¥ISATS¥¥WISnaD> 1MQFLTSLAAAASLVSLASAftlSGIALPQTVKAGDNINAIVVTEGYIQSVQDIAIAFGCAPAA SAYPGTLSTLLGSFYLGPEQCNVQNNITEPITIPESLVPGEYVIAASLFSLYGASSSPTVSN I lYNVTVNVGNET^ETYVRSQFCVGNSNSTVGLGGYTRKINALSGT PCX Intein I CH Methylation Degradation Phoshphorylation 16 Sample identity MS identification 1 MKKESINTSG 51 GVGTNNAVWH 101 RGTDNALWHN 151 DNALWHIWQT 201 SLWYIKQTAS 251 WHIWQVAPNA 301 WQTATSDAWS 351 TSSWSTWTSL PDNTKSSISD NWQTVPNTGS WQTVPGAGWS APHAGPWSNW HTYPWTNWQS GWTNWRSLSG EWTSLSGVTT GGNLIDASAI MS intact mass analysis 10000 20000 30000 40000 50(KiO EIEISNEISW SWSGWHSLNE GWQSLGGQIT QSLNGVLTSD LSGVITSNPV IITSDPAVHI SAPTVAKNSD K TALSGVISAA GATSKPAVHI SNPWYINSD PTVYVNASGR VISNSDGRLE NADGRLEVFA GWLEVFARGA NNADGRLEVF NSDGRLEVFV GRLEVFARGA PEVFARSNDY VFARGSDNAL RGPDNALWHI NNALCHIQQT Post-translational modifications Isotope labeling Matrix adducts m/z 17 Sample purity Contaminants - co-purified molecules Small molecules . Macromolecules • Co-factors . Protein isoforms • Ligands . Proteins • Salts, imidazole . Nucleic acids • Lipids • Polysaccharides • Saccharides . Binding partners Sample purity - methods SDS-PAGE • UV-VIS spectroscopy • SEC (SEC-MALS) • FFF (FFF-MALS) • Mass spectrometry small molecules Co-factors Ligands Salts, imidazole Lipids Saccharides macromolecules Protein isoforms Proteins Nucleic acids Polysaccharides Binding partners SDS-PAGE • Polyacrylamide gel (8 - 20 %) • SDS - uniform (?) protein charge (composition dependent) • Reducing agent (optional) - (3ME • Staining - CBB, Silver, Fluorescent, Radiological Coomassie staining Silver staining Fluorescent protein staining Sensitivity 5-25 ng 0.25-0.5 ng 0.25-0 5 ng SDS-PAGE • Check overloaded as well as underloaded sample ^ r / o1 ^ UV-VIS spectroscopy (200-) 240-340 nm Trp (and Tyr) has absorption peak around 280 nm • Detection of: • Nucleic acid contamination • Aggregation (scattering) • UV-absorbing contaminants Nucleic acids arbre-mobieu.eu 280 300 Wavelength nm 370 340 240 260 290 500 22 Size exclusion chromatography • Separation of particles based on "size" • Interaction with matrix possible (!) • Possibility to couple to multiple detectors (UV, Rl, MALS, viscosity) Elution volume (ml) Field flow fractionation Separation of particles in solution by external force Field Flow Fractionation Outlet to the detector Separation field Parabolic flow profile Diffusion Separation field Field of force Techniques r Thermal -1- Centrifugal ThFFF Flow I T Electrical ^^^^^^ 1 SdFFFor CFFF Magnetical FIFFF (SF4) EIFFF MgFFF AF4 CEIFFF HF5 24 Mass spectrometry • Detecting of exact mass of particles • Various applications based on set-up Sample homogeneity • Macroscopic - precipitation - visual detection • Microscopic - oligomeric states, folding states, microheterogeneity - biophysical methods Sample homogeneity vs. purity Various methods may evaluate sample in different way o o o oo o o • Homogenous Good sample Sample homogeneity - methods • SEC-MALS, FFF • Native electrophoresis Light scattering Analytical ultracentrifuge 28 Native electrophoresis • Possibility to observe various oligomers (relatively imprecise and unreliable) and isoforms (2D PAGE preferred) • Not efficient for aggregation detection Light scattering • Interaction of incident light with particles in solution • Intensity of light at given • Typically red/infrared light Light scattering • Dynamic light scattering - size of particles - sensitive to aggregation • Static light scattering - mass of particles - averaged value, separation required (a) IM (b) 100ftJnmt*Al*y Large Particles oo Small Particles o o oo o arbre-mobieu.eu IM C(q.T) Log(r) _« -.^A tkA Log(r) Static light scattering (SLS) Low-angle light scattering (LALS) - big molecules Right-angle light scattering (RALS) - small molecules Multi-angle light scattering (MALS) - Mw and Rg • Intensity of scattered light • Mass of the particle (molecular weight) Static light scattering Average of all sample particles ! Typically coupled to separation (SEC, FFF) ^10x10 on 4 £ 1.0x10 O 1000.0 MALb dRI Hexamer 35 kDa / \ Monomer v / \ 5.8 kDa V I y SEC-MALS 13.0 14.0 15.0 16.0 17.0 volume {ml_) 18.0 16 17 Retention Time [min) Dynamic light scattering (DLS) Time-dependent fluctuations in scattered light Size of the particle (hydrodynamic radius) Laser Sample «: £ 500 c H fff/m - 400 - i f f > /'' I 300 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 radius (cm) polydisperse sample 5 10 15 20 sedimentation coefficient (S) 5 10 15 20 sedimentation coefficient (S) 25 AUC - Sedimentation equilibrium • Distribution of particles in cell • Molecular mass of particle • Problematic for mixtures Desai 2016 radiUS (cm) www.nanolytics.de Comparison Light scattering Analytical ultracentrifugation Sample volume 0.5-30 ul (DLS) 1-50 ul (SLS, SEC-MALS) 150-450 ul Sample concentration 0.1-200 mg/ml 0.1-1 mg/ml Particle size 1 nm-10 Lim 1-300 nm Resolution and accuracy Low - Average Average - High Speed of analysis 1 min (DLS, SLS) 30 mins (SEC-MALS) 4 hrs (SV) 3-4 days (SE) Sample stability • Temperature stability • Chemical stability • pH • Ionic strength • Oxidizing agents • Protein-specific compounds • Long-term stability - storage Temperature Affects stability and interaction parameters i,^ iy R(t) Arrhenius equation In KA = pQ k= Ae v Typical temperatures: -80 °C, -20 °C, 4 °C, 20 °C, 25 °C, 37 °C Room temperature (RT) - vaguely defined mostly 20 - 25 °C, but varies from 15 - 30 °C usually means that temperature was not set (!) 42 pH=-log[H30+] Typical range: 4-9, specific proteins 1-12 pH of pure water: 7 (theor.), 5.8 (due C02 absorption) Buffers: dissociable compounds with defined pKa various pH ranges - typically (pK -1) - (pK_+l) pH - buffers • Organic/Inorganic • Universal buffers -mixtures with broad pH range aims Biophysics Volume 2, Issue 3, 336-342. DOI: 10.3934/biophy .2015.3.336 Received dale 19 April 2015, Accepted date 20 July 2015, Published date 14 August 2015 http://www.aimspress.com/ Letter Universal buffers for use in biochemistry and biophysical experiments Dewey Brooke \ Vtxid Movahed \ and Brian Bothner * Department of Chemistrs and ISioehcmistrv. Montana State I niversitv Uozeman M I 59717. I SA Good's Buffer pKa(20 °C) PH MES 6.15 5.5-7.0 Bis-Tris 6.46 5.7-7.3 ADA 6.60 5.8-7.4 PIPES 6.80 6.1-7.5 ACES 6.90 6.0-7.5 MOPSO 6.95 6.2-7.4 BES 7.15 6.6-8.0 MOPS 7.20 6.5-7.9 TES 7.50 6.8-8.2 HEPES 7.55 6.8-8.2 TAPSO 7.70 7.0-8.2 POPSO 7.85 7.2-8.5 HEPPSO 7.90 7.4-8.6 EPPS 8.00 7.5-8.5 Tricine 8.15 7.8-8.8 Bicine 8.35 7.7-9.1 TAPS 8.40 7.7-9.1 CHES 9.50 8.6-10.0 CAPS 10.40 9.7-11.1 44 PH Ionic strength Ionic strength, /, is a measure of the concentration of electrically charged species in solution i Square root of ionic sirenglu Protein solubility changes with ionic strength as well as with solute composition Impurities/Additives OH • Various compounds affect protein stability/solubility H0^ o,ov X ^oh HO°LS^J"0 °> H OH H C0H • Saccharides - saccharose, trehalose • Amino acids- Arg, Glu, Pro nh o • Reducing/oxidizing agents - pME, DTT, TCEP h2n^n^-^y^oh DMSO NH- Protein-specific compounds (ligands) 0 OH HS O 6h ho^p^oh X H3C^ VCH3 Buffer optimization • Buffer affects: • Stability • Activity (interactions) • Storage • Many buffers do not meet all requirements Buffer optimization desired Buffer optimization Various commercial screens available Differences in composition, number of conditions Example: buffer screen designed by CF BIC, CEITEC MU 2 3 5 6 8 9 10 11 12 I H2° pH 2-12 pH 4-9.5 (different buffers to those in A row) Ionic strength (for pH 6-8) Pre-defined buffers Additives 49 Buffer optimization n 2 3 4 5 6 7 8 9 10 11 12 59.2°C 43.6°C 37.7°C 55.0°C 61.3°C 59.8°C 62.1°C 55.5°C 59.0°C 33.4°C 33.2°C B 36.5°C 42.1°C 48.3°C 52.2°C 55.0°C 58.5°C 66.2°C 66.4°C 66.5°C 58.7°C 59.4°C 63.1°C 63.3°C C 57.2°C 59.2°C 60.6°C 58.5°C 62.7°C 62.1°C 67.0°C 68.1°C 69.9°C 60.2°C 61.8°C 66.5°C 70.0°C D 69.4°C 63.4°C 46.2°C 55.2°C 58.2°C 54.5°C 59.2°C 59.5°C - 59.2°C Buffer screen C7 + C12 condition Sample storage Depends on sample stability Freezing (phase transition) may decrease protein stability in solution Avoid repeated freeze-thaw cycles ! Fridge: 4 °C Freezer: - 20 °C, - 80 °C (cryo-protectants addition - glycerol) Lyophylization = Freeze-drying: water sublimation Check sample quality BEFORE and AFTER storage ! 51 Batch to batch quality check Enormous amount of variables in preparation process Two sample batches may not be the same Minimal tests desired to verify sample quality 52 Reproducibility crisis Based on 2016 poll with > 1500 scientists included: 70 % were not able to repeat an experiment! 50 % were not able to repeat at least one of their own experiments !!! Possible causes: • Selective choice of data (cherry picking) • Unsuitable experimental desing • Inappropriate data evalueation (statistics) It's probable that partial problem is insufficient characterization of input material and procedures. Source: nature.com 53 Summary • Sample quality is crucial for downstream experiments • Various sample properties to be checked • Identity • Purity • Homogeneity • Stability Storage and buffer optimization desired 54 Questions? 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