Methods in Biochemistry Metodologie Biochimiche Anno 2018-19 Stefania Brocca, Matilde Forcella, Paola Fusi [email protected] Tel. int.: 02 6448 3518 room: 5054, U3 - 5th floor https://elearning.unimib.it/ Metodologie Biochimiche 2018-19 01) Martedì 2/10 02) Venerdì 5/10 03) Martedì 9/10 Modulo frontale 04) Venerdì 12/10 Esercitazioni 4 crediti 05) Martedì 16/10 2 crediti (14 lezioni) 06) Venerdì 19/10 (20 h in 5 pomeriggi) 07) Martedì 23/10 Aule: U1-04; U1-10 08) Venerdì 26/10 3 turni da 1 settimana 09) Martedì 30/10 dicembre 2018, gennaio 2019, 10) Martedì 06/11 febbraio 2019 11) Venerdì 09/11 Lab 1026 12) Martedì 13/11 13) Venerdì 16/11 14) Martedì 20/11 Program (theoretical lessons) Protein «environments» Electrophoresis Hydrophobic effect Principles Solubility, salting in/salting out SDS-PAGE Hofmeister series (Blue)-native gel Denaturing agents Band-shift assay Dialysis Isoelectrophocusing Centrifugation techniques Bidimensional gel Principles Western blot Differential centrifugation Spectroscopies Density gradient centrifugation Principles Analytical ultracentrifugation Absorbance Chromatography Fluorescence Principles Circular dichroism Chromatographer set up Mass spectrometry Gel filtration Surface plasmon resonance Ion exchange Hydrophobic interactions Affinity tags Suggested references Wilson K. & Walker J. (2000) “Biochimica e Biologia Molecolare” Cortina, 2006 Numero di pagine: 777 Prezzo: ~ € 100 M. C. Bonaccorsi di Patti, R. Contestabile, M. L. Di Salvo Cited articles “Metodologie Biochimiche” Casa Editrice Ambrosiana, 2012 Numero di pagine: 289 Prezzo: ~ € 40 Protein preparation and analyses Cells/tissues Lysis Purification Characterization Protein extraction & purification Analitycal techniques (titration assays, structual analyses etc) Lessons 1-2 • Structure and stability of proteins • Protein denaturation • Hydrophobic effect • Ion solvatation • Protein solvatation • Hofmeister series • Protein precipitation-dialysis • Kinds of buffers Structure and stability of proteins Many factors can damage proteins • Temperature • Pressure • pH • Proteolytic activity • Reactive oxygen species (ROS) • Organic solvents • Urea/Guanidinium • Tannins Aim: keep protein native conditions Protein structure is stabilized by weak interactions • Hydrogen bonds • Van der Waals interactions • Electrostatic interactions • Hydrophobic effects It is not a direct attraction, but an «apparent force» that stems from entropy and involves solvent molecules Thermodynamics in protein folding ΔG = ΔH - TΔS Net difference of stability (∆G < 0) Hydrophobic effect (-T∆S << 0) In physiological conditions, ΔG <0. Low value! H bonds (∆H << 0) Conformational entropy Van der Waals of polypeptide chain interactions (∆H << 0) (-T∆S >>0) Proteins can be easily denatured!!! https://employees.csbsju.edu/hjakubowski/classes/ch331/protstructure/olhydrophobprot.html Hydrophobic or solvophobic effect • Hydrophobic effect: apolar compounds tend to aggregate in acqueus medium. Solvophobic effect applies to polar solvents different from water • The exclusion of non-polar groups from water is energetically favoured, (ΔG <0). Indeed, around non-polar groups, water molecules are more ordered (ΔS <0) water water Organic molecules One «hole» in water is better than two! ….. ….. Hydrophobic interactions and water structuring Native form Denatured form “quasifrozen” water patches about nonpolar molecules Frank and Evans, (1945) The journal of chemical physics. Hydrophobic interactions and water structuring Benzene «clathrates» Phe Hydrophobic effect lowers with temperature The transfer of a nonpolar solute into The transfer into hot water cold water (e.g. room temperature) is (e.g., boiling point) is opposed opposed by a large entropy. by a large enthalpy (needed to Dill et al., (2005). DOI:10.1146/annurev.biophys.34.040204.144517 boil water). Hydrophobic effect Summing up • Hydrophobic effect occurs when apolar compounds tend to aggregate in acqueus medium • Solvofobic effect is a hydrophobic effect occurring in a polar medium different from water • It is due to an «apparent» force (not direct attaction) • Entropic origin (exposition of apolar groups to water causes an ordered set-up among solvent molecules) Salt solutions Ionic strength (I ) 푛 1 Measure of charge 퐼 = 푐 푧 2 concentration in solution 2 푖 푖 푖=1 c = concentration, z = charge Es. NaCl 1 M I = 1/2 (1 . 12 + 1 . 12) = 2/2 = 1 . 2 . 2 Es. MgCl2 1 M I = 1/2 (1 2 + 2 1 ) = 6/2 = 3 • Salts affect both protein solubility and conformational stability • These effects depend on the kind of salt • Some salts can stabilize, others can denature proteins Water ordering Li+ and ionic radius - F Small Na+ + - Large Water K Cl Rb+ Br- Cs+ I- Water ordering depends on the ionic radius Bulk 2nd hydratation shell 1st hydratation shell Na+ Smaller the radius, thicker the shell Water ordering Li+ and ionic radius - F Small Na+ (kosmotropic ions) Large K+ Cl- Water (kaotropic ions) Rb+ Br- Cs+ I- Water ordering depends on the ionic radius Kosmotropic ions (ionic radius) Chaotropic ions For a given ionic radius, a negative ion interacts more strongly with water than a positive ion does The mystery of charge asimmetry - small ions - water water one H pointing outside F- Li+ More water dipoles and more both H pointing outside closer around the anion The mystery of charge asimmetry water - large ions - water Low charge density I- Cs+ • Weakly hydrated ions (bulk water–water bonds favored) • Tetrahedral water structures destabilized in their vicinity hydrophobic effect!! Larger ions are less hydrophilic Organization of water around a protein >ns ~ps protein surface inner How do salts affect protein solubility? Solubility of carboxyhemoglobin solubility in various electrolytes at 25 °C of Logarithm Salis e Ninham , DOI: 10.1039/C4CS00144C , Adattato da Green, J. Biol. Chem., 1932, 95 Root square of ion strength Low salt concentrations always favour solubility High salt concentrations can have different effects Poor solubility Salting in Salting out salt-solvent interactions Salting in Unfolding At low concentrationSalt concentration Protein-protein interactions salt-protein interactions At low concentration, salt screens out At concentr. > 0.1 M, salt competes for protein-protein charge interactions. water and removes the solvation sphere. Higher solubility Salting in “Salting out” or unfolding? Franz Hofmeister (1850-1922). First Faculty of Medicine, Charles University in Prague How do salts affect protein solubility? At salt concentration >0.1 M, certain kind of ions cause proteins precipitate out of solution (i.e., are salted out). Other types of salts cause proteins to become more soluble (salted in). Hofmeister series: ranking of ions for their ability to solubilize or precipitate proteins • Strong solvent interactions • Strong protein interactions • High interfacial tension • Lower interfacial tension • Native proteins precipitate due to • Proteins solubilized by interaction unavailability of solvent. Stabilizers with solvents. Denaturants Direct Hofmeister series Strong solvent Ion radius interactions ok Strong interactions Ion radius with protein… …depending on protein charges Electrostatic surface potential of a protein negative positive Direct Hofmeister series “Direct series”: empirically observed for proteins with mixed apolar and negatively charged surfaces “Indirect series”: reversed effects observed for proteins with mixed apolar and positively charged surfaces Isoelectric point (pI) = pH is the pH at which a molecule carries no net electrical charge in the statistical mean pI 10 pI 9 Protein A Protein B Electrostatic surface potentials of a protein pH 4.0 pH 7.0 pH 11.0 Several diverse Hofmeister series!! negative positive Electrostatic surface potential of three homologous proteinases K negative positive Helland et al. (2006) FEBS Journal 273:61–71 Schwierz et al. (2016), https://doi.org/10.1016/j.cocis.2016.04.003 Let’s start with some fixed points…. Guanidium chloride Tetramethylammonium Chaotropic ions = denaturing agents Guanidinium and other denaturants that lower solvophobic effect guanidinium (6 M) urea (8 M) Usually don’t cause Acetonitrile, methanol, ethanol, propanol etc aggregation Ammonium sulfate • Has a wide range of application • Proteins loose solubility as amm. sulfate concentration increases • Each protein precipitates at a particular concentration (empirically determined) Partial purification of all proteins with similar solubility characteristics. “Salt cuts”: fractionation of proteins from the same solution /extract Large-volume preparations Protein concentration (first steps of purification) Each protein precipitates at a particular ammonium sulfate concentration Protein response is specific and depends on composition, pI, size, quaternary structure etc. Scheme of «ammonium sulfate cut» 50% saturation ammonium sulfate (AS) pellet (contaminants) centrifugation supernatant 70% saturation AS pellet (target protein) centrifugation supernatant Ammonium sulfate precipitation table Grams for 1 L solution Final concentration (in % of solution saturation) concentration Starting Dialysis • Passage of solutes through a semi- permeable membrane. • Size of pores is defined in terms of cut-off (MW under which a solute can cross the membrane). • Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through http://zoology.okstate.edu/zoo_lrc/biol1114/tutorials/Flash/Osmosis_Animation.htm Animation Protein minimal solubility is at pH = pI Aggregation at pH = pI Dialysis Dialysis is usually applied to buffer exchange. At the end of the procedure, the external buffer will substitute the internal one (internal components go outside and VICEVERSA). Video-procedure dialysis: http://www.youtube.com/watch?v=2Th0PuORsWY http://www.youtube.com/watch?v=y0ffJBFQswk http://www.youtube.com/watch?v=0Km2C2IwPLI .