Protein Characterisation Lecture - PDF

Molecules Genes & Cells Exploring Proteins Protein Characterisation Iain McNae [email protected]  A new protein Absorbance Sobole...

Molecules Genes & Cells Exploring Proteins Protein Characterisation Iain McNae [email protected]  A new protein Absorbance Sobolevsky et al., Nature, 2009 Is the protein composed of one or more polypeptide chains? What mass is each polypeptide chain and the whole protein? SDS-PAGE, mass spectroscopy (protein denatured) Size-exclusion chromatography, ultracentrifugation, light scattering (in solution, protein NOT denatured) What is the surface charge of the protein? Isoelectric focusing (protein denatured)  SDS-PAGE to determine molecular mass of a protein  Native-PAGE Protein not denatured (no SDS) and disulphide bridges not reduced (no reducing agent). → Protein retains its tertiary (and quaternary) structure Coomassie Brilliant Blue G-250 -imparts neg. charge w/o unfolding: M  Combining SDS-PAGE and Native PAGE. SDS-PAGE gel: NATIVE gel: Ge, Nature, 2015 Single band on SDS-PAGE and single band on Native PAGE It‘s a homotrimer - it is made of 3 identical subunits (polypeptide chains).  Mass Spectrometry Results mass / charge ratio (m/z) All in vacuum. MALDI - Matrix-assisted laser Protein is denatured and not desorption/ionization recovered. TOF – Time of Flight  Analytical Ultracentrifugation Sand sediments in a glass of water (Soluble) proteins don’t …due to gravity (g = 9.821 ms-2). …due to their thermal (Brownian) motion which keeps them evenly distributed throughout the solution. → Proteins will start to sediment like sand grains only when subjected to enormous acceleration (e.g. 600 000 x g).  Analytical Ultracentrifugation Sedimentation coefficient (s) for a protein: 𝑚 𝑥 (1 −⊽𝜌) S= [S = svedberg] 𝑓 Mass m = protein mass ⊽ = partial specific volume of protein Shape 𝜌 = solution density f = frictional coeff of protein  Light Scattering Light after passing through the sample Incoming light I Io Purified protein  Light Scattering Laser in (incoming light) Detectors at 2 Protein Scattered different angles (macromolecule) light Images from http://www.wyatt.com/theory/rayleighscattering/mass.html Light scattered from different parts of the protein will have different intensities This is due to constructive and destructive interference of the scattered light Intensity of scattered light will vary with angle 𝜃  Light Scattering Elastic or Rayleigh scattering (no change in wavelength) http://www.lsinstruments.ch/technology/static_light_scattering_sls/ Multiangle Light Scattering (MALS)  Light Scattering and Molecular Mass Determination Theory: Iscattered light ∼ protein concentration x M Combine size-exclusion chromatography and light scattering (SEC-MALS): Direct read-out of protein SEC column mass per fraction. Laser beam MALS detectors  Spectroscopic techniques Light after passing through Purified protein the sample Incoming light I Io Wavelength matters! Interaction with sample results in a change to emitted light. Which we measure.  Spectroscopic techniques m X-ray crystallography Fluorescence Absorption Nuclear Magnetic Resonance Circular Dichroism  Protein Absorption Spectroscopy Planck‘s constant = 6.626 x 10-34 J*s Frequency = c/𝜆 E=hν E - - - - - + - - Energy matches–transition E=hν http://wpcontent.answcdn.com/wikipedia/commons/thumb/a/a9/Amino_Acids.svg/440px-Amino_Acids.svg.png  Protein Absorption Spectroscopy Phe Tyr Trp 280 nm E = 7*10-19 J By NEUROtiker - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=1637099 Protein Absorption Spectroscopy l = 1 cm Incoming light Io I A = log10 (Io/I) A = ecl l = light pathlength (cuvette width) = 1 cm c = protein concentration (unknown) 𝜀 = molar extinction coeff of a protein (literature)  Protein Concentration A = ecl Amino Acid lmax (nm) e ( M-1cm-1) W Tryptophan 280 5 600 Y Tyrosine 274 1 400 F Phenylalanine 257 200 H Histidine 211 5 900 Adenosine 260 13 400 Cytosine 267 6 100 Circular Dichroism Circular Dichroism  Circular Dichroism Circularly polarized light: L (left) and R (right) propagates down the axis of direction cddemo.szialab.org Differential Absorption of L and R Circularly Polarised Light A = eLcl A = eRcl Measure: eL – eR = (AL/ eLcl - AR/ eRcl)  Circular Dichroism CD – binding of paracetamol to Human Serum Albumin. eL – eR Can give a measure for percentage of secondary structure types. Fluorescence Spectroscopy Fluorescence  Fluorescence in a nutshell Absorbance Slow Emission Shorter fast fast Longer Wavelength Shorter Wavelength Longer Wavelength - Wavelength Incoming light results in higher Two stage return has less energy energy state resulting in lower energy emission Absorbance Emission Wavelength  Fluorescence Absorbance Slow Emission fast fast Shorter Longer Wavelength - Wavelength Fluorescence of Tryptophan Abs Emission  Binding of paracetamol to human serum albumin W - Fluorescence Paracetamol increases END