4D structure Josef Houser Autumn 2023 S1004 Methods for structural characterization of biomolecules Structural hierarchy primary (sequence) secondary tertiaryquaternary ADSQTSSNRAGEFSIPPNTDFRAIFFANAAE QQHIKLFIGDSQEPAAYHKLTTRDGPREATL NSGNGKIRFEVSVNGKPSATDARLAPINGK KSDGSPFTVNFGIVVSEDGHDSDYNDGIVV LQWPIG 2D1D • Association of individual (protein) chains • Consisting of identical chains (homooligomers) or different chains (heterooligomers), including non-protein molecules, e.g. nucleic acids 3 Quaternary structure • Composition of the complex molecule may be obtained: • Via dedicated experiment (MS, SEC-MALS, ...) • From 3D structure • Combination of both • Composition depends on conditions 4 Quaternary structure Concentration-dependent • Dimerization – special type of protein interaction • KD(dimerization) can be determined – interaction techniques • At c < KD → dimer not stable !!! 5 KD Sample homogeneity vs. oligomerization • Working concentration determines protein state 6 Monomer AggregateDimer Oligomer c(protein) Stability methods • Indirect evidence – double (multiple) transitions upon heating • Thermal-shift assay Differential scanning fluorimetry • Advantage: easy-to-obtain data • Disadvantage: additional knowledge needed; risk of misinterpretation 7 Native Mass spectrometry (MS) • High precision in MW determination • Easier for stable oligomers – S-S bonds • Mild ionization – ESI-MS 8S.Tamara et al, Chem. Rev. 2022 Native electrophoresis • Possibility to observe various oligomers • Relatively imprecise and unreliable • Complicated in presence of protein isoforms 9 Pandhare 2019 arbre-mobieu.eu Size exclusion chromatography • Separation of particles based on “size” • Interaction with matrix possible (!) Preparative Analytical Bigger volume “Minimal” volume Long runs (hrs) Short runs (minutes) Peak separation required Peak separation advantageous 10 Field flow fractionation • Separation of particles in solution by external force • Alternative to SEC 11 Size exclusion chromatography • Single detector (UV) • MW determination depends on standard elution volume • Non-standard behavior = MW error • Multiple detectors (detector array) • Precise concentration (RI, ev. UV) • Accurate MW (MALS, RALS/LALS) 12 Light scattering • Interaction of incident light with particles in solution • Intensity of light at given angle • Typically red/infrared light 13 • Dynamic light scattering – size of particles – sensitive to aggregation • Static light scattering – mass of particles 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) www.malvernpanalytical.com LALS RALS MALS 1/Mw 14 Static light scattering • Average of all sample particles ! • Typically coupled to separation (SEC, FFF) 15 Analytical ultracentrifugation (AUC) • Sedimentation of particles in centrifugal field by hydrodynamic properties • Two modes: • Sedimentation equilibrium – mass determination • Sedimentation velocity – size distribution xenon flash lamp diffraction grading incident light detector photomultiplier tube cell slit movable imaging system AUC – Sedimentation velocity • Sedimentation of particles over time observed • Size of the particle (hydrodynamic radius) AUC – Sedimentation velocity • Suiteable to detect and quantify oligomers and aggregates • Sensitive to shape (and density) 18 AUC – Sedimentation equilibrium • Distribution of particles in cell • Molecular mass of particle • Problematic for mixtures – homogeneity !!! Desai 2016 www.nanolytics.de Mass photometry (MS) • Measurement of scattered light interference • Mass of the particle 20 Mass photometry (MS) – instruments • Single producer • Manual and automated version • Low volume at low sample concentration 21 Small-angle X-ray scattering (SAXS) • Size and shape of molecules • More in a separate lecture by Tomáš Klumpler 22 Summary • Oligomeric state depends on conditions (concentration) • Various techniques with different requirements • Choose technique(s) suitable for your system • Be aware of results misinterpretation 23 Questions? 24 ? 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