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PC 204 Biophysical Methods

PC 204 Biophysical Methods. Katalin F. Medzihradszky. Biophysical Methods. Complement X-ray and NMR structural information Measure how proteins behave in solution Can determine how they dynamically interact with each other Can determine how they interact with different substrates

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PC 204 Biophysical Methods

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  1. PC 204 Biophysical Methods Katalin F. Medzihradszky

  2. Biophysical Methods • Complement X-ray and NMR structural information • Measure how proteins behave in solution • Can determine how they dynamically interact with each other • Can determine how they interact with different substrates • Can make measurements in situ • Can provide mapping information

  3. Major Techniques • Hydrodynamic methods (analytical ultracentrifugation, viscometry, etc) • Thermodynamic methods (light scattering, microcalorimetry, surface plasma resonance, etc) • Spectroscopy (fluorescence, circular dichroism, electron paramagnetism, etc) Do NOT write off the old techniques!

  4. Courtesy of Balázs Szalontai www.brc.hu

  5. Interaction of light with the molecules Electronic excited states Vibrational levels Rayleigh Raman Visible absorption Fluorescence Resonance Raman Infrared absorption Vibrational levels Electronic ground states Courtesy of Balázs Szalontai www.brc.hu

  6. An imaginary view of the biological membrane Source: http://www.ncnr.nist.gov/programs/reflect/cnbt/

  7. Infrared spectrum of a thylakoid membrane in D2O 3 (H-O-D) +( N-D) proteins proteins D2O lipids 2 lipids Absorbance (OD) D2O 1 lipids 0 4000 3500 3000 2500 2000 1500 1000 Frequency (cm-1) Courtesy of Balázs Szalontai www.brc.hu

  8. FT-IR can be used for detecting changes in the membrane, eg within protein-lipid interactions; protein structure etc. amid II amid I C=O Structure-sensitive vibrations in biological membranes

  9. Presley JF, Biochimica et Biophysica Acta 1744, 259-272 (2005)

  10. Presley JF, Biochimica et Biophysica Acta 1744, 259-272 (2005)

  11. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in chemistry for their discovery and development of the green fluorescent protein. (A) The wtGFP chromophore, consisting of a cyclized tripeptide made of Ser65, Tyr66, and Gly67. (B) Chromophore location within an a  helix inside the 11-strand b barrel of GFP. (C) wtGFP's major and minor absorbance peaks (blue circles) and single fluorescence emission peak (green circles) compared with EGFP's single absorbance peak (blue squares) and single fluorescence emission peak (green squares). Science Lippincott-Schwartz et al., pp. 87 - 91

  12. GFP major classes • Wild type • EGFP (phenolate anion) • H9 (neutral phenol) • Yellow flourescent protein (phenolate anion/pi electron system) • Cyan flourescent protein (indole in chromophore) • Blue flourescent protein (imidazole in chromophore) • - (phenyl in chromophore)

  13. A) Excitation and B) emission spectra of some commonly used fluorescent proteins including the blue fluorescent protein (BFP), cyan fluorescent protein (CFP), GFP, and YFP derived from the jellyfish Aequorea victoria GFP, the (tetrameric) red fluorescent protein from the coral Discosoma sp. (DsRed), the far red fluorescent protein from the sea anemone Entacmaea quadricolora (eqFP611), the monomeric red fluorescent protein derived from DsRed (mRFP1), and the (dimeric) far red fluorescent protein derived from the purple chromoprotein of Heteractis crispa (HcRed1). Schultz C et al, Chembiochem 6, 1323-30 (2005)

  14. Watch this!Live imaging of dense-core vesicles in primary cultured hippocampal neurons.Kwinter D, Michael S.J Vis Exp. 2009 May 29;(27). pii: 1144. doi: 10.3791/1144. ~10 min video

  15. A pulse–escape fluorescence photoactivation experiment. Example of a wild-type DRG neuron that was transfected with PAGFP-NFM and a diffusible red fluorescent protein (DsRed2) as a marker for transfection. A, The DsRed2 fluorescence, which fills the axon. B, A preactivation image of the same axon showing no GFP fluorescence. C, An image of the same axon immediately after activation using violet light. D, An image of the same axon 2 h later. The white arrowheads show the location of the activated region. The decrease in fluorescence between C and D is attributable to departure of fluorescent neurofilaments from the activated region, but note that many neurofilaments remain. Scale bar, 5 µm. Tracking the movements of neurofilaments J Neurosci. 2009 May 20;29(20):6625-34. Myosin Va increases the efficiency of neurofilament transport by decreasing the duration of long-term pauses. Alami NH, Jung P, Brown A.

  16. Translocation probes permit monitoring of concentration changes of molecules of interest in different subcellular compartments. Commonly used probes attach to the plasma membrane by recognizing a lipid species, in this case diacylglycerol (DAG), by using the C1a domain of PKC. HeLa cells were transfected with YFP-C1a and the bradykinin receptor (BK2), and were stimulated with 1 M bradykinin. This activated phospholipase C (PLC) as indicated by the translocation of the DAG-binding domain from the cytosol (t=0 s) to the plasma membrane (t=63 s). The formation of DAG by PLC was analyzed over time by quantifying cytosolic fluorescence (see graph). A fast increase in DAG was followed by a slow turnover of the DAG signal. ChemBioChem Vol.6, 8 Pages: 1323-1330

  17. FRET = Forester (or fluorescence) Resonance Energy TransferFLIM = Flourescence Life-time Imaging Microscopy

  18. FRET • Requires donors and acceptors (D & A) • Components (bearing fluorescent probes) must come in close enough contact so that non-radiative energy transfer can occur. • The two fluorophores have sufficiently large spectral overlap, a favorable dipole-dipole orientation, proximity of 1-10 nm and a large enough quantum yield. • Can be measured with confocal or multiphoton microscopy

  19. FLIM • Technically more challenging than FRET • Measures the rate of decay of the emission’ which is a property of the fluorophore • Unaffected by probe concentration or excitation intensity • It is affected by environment, eg change in pH, and by FRET • Can make combined FLIM-FRET measurements by monitoring the change in donor lifetime in the presence and absence of acceptor.

  20. Wallrabe H and Periasamy, A Curr Opin Biotechnol 16, 19-27 (2005)

  21. Designs and applications of fluorescent protein-based biosensors.Ibraheem A, Campbell RE. Curr Opin Chem Biol. 2009 Nov 11. [Epub ahead of print]

  22. Biosensors • Biosensors = devices that transduce a biorecognition event, such as an antibody-antigen binding or the formation of a DNA duplex, into a measurable electronic or opto-electronic signals Fan et al Trends Biotech 23, 186-192 (2005)

  23. FRET-based biosensor designs. (a) Schematic model of a generic intramolecular FRET-based biosensor. A FP FRET pair flanks an MRE that undergoes a conformational change that alters the distance and/or orientation of the FPs relative to each other. (b) An MRE suitable for the detection of protease activity. (c) An MRE for the detection of PTM enzymatic activities where the modification of the peptide substrate creates a binding dock for the binding domain resulting in a FRET change. (d) An MRE in which the conformational change is triggered by the presence of its analyte. (e) Protein–protein interactions can be visualized in live cells by tagging each one of the proteins to one member of a FP FRET pair and observing the changes in donor/acceptor intensities. MRE = molecular recognition element

  24. A) Spectra of the recombinant PKC-FRET probe, KCP-1, before and after phosphorylation by a soluble catalytic subunit of PKC. Specific phosphorylation of the substrate loop by PKC leads to an increase in FRET. The donor is GFP2; the acceptor is enhanced YFP (EYFP). B) PKC activity monitored over time in N1E-115 cells that are transiently transfected with KCP-1 and stimulated with bradykinin and phorbol ester (TPA).

  25. Additional designs of FP-based biosensors. (a) Detection of a protein–protein interaction by BiFC. (b) Single FP biosensors with an exogenous MRE. (c) Single FP biosensors with an endogenous MRE. (d) A hybrid design that used a pH-sensitive acceptor fluorophore in a FRET pair.

  26. Toward visualization of nanomachines in their native cellular environment.Pierson J, Sani M, Tomova C, Godsave S, Peters PJ.Histochem Cell Biol.132(3):253-62 (2009).

  27. Cryo-electron-tomography (ET) • ET is used to generate 3D maps from a series of 2D transmission electron microscopy images. The specimen is rotated with respect to the imaging source • Cryo-ET is based on a freezing technique that captures the cellular water in an amorphous (glass-like) layer in which all cellular components are embedded. Single particle analysis – analysis from large number of purified macromolecular particles: One has to identify individual particles within the micrograph, they have to be properly aligned based on characteristic recognition features (larger complexes are easier to handle); then the images are averaged  build 3D structure • Only symmetrical complexes can be studied, except the ribosome • These images could be utilized to localize the corresponding complexes within the cell

  28. From a whole cell to isolated complexes. a Electron micrographs of osmotically shocked S. flexneri exhibiting the type III secretion system protruding through the bacterial envelope (see boxed area). b Gel chromatography of solubilized secretion complexes from the bacterial envelope. c The fraction containing enriched complex is checked by SDS and individual bands identified by mass spectrometry. d2D projection average of 1,500 isolated needle complexes composed of a hollow needle appendage (indicated by a stain-penetrated line along its axis) and a basal part of several rings that traverse the cell envelope. The channel that runs through the needle appendage is 2–3 nm in diameter indicating that substrates that exit the conduit do so in an unfolded state

  29. Soft X-ray tomography and cryogenic light microscopy: the cool combination in cellular imaging. McDermott G, Le Gros MA, Knoechel CG, Uchida M, Larabell CA. Trends Cell Biol.19(11):587-95 (2009).

  30. Soft X-ray microscopy • Advantages over light and electron microscopes. i) significantly higher spatial resolution because of the short wavelength. ii) fully hydrated specimens up to 10 μm thick can be imaged without the need for sectioning iii) image contrast is obtained directly from the absorption of X-rays by the specimen. • Wavelength used within the “water window”. The water window is the region of the spectrum that lies between the K shell absorption edges of carbon (284 eV, λ=4.4 nm) and oxygen (543 eV, λ=2.3 nm). Photons within this energy range are absorbed an order of magnitude more strongly by carbon- and nitrogen-containing organic material than by water.This absorption is linear with thickness and concentration. • Consequently, images produced by soft X-ray microscopes are quantitative, with each biochemical component having a signature X-ray linear absorption coefficient (LAC). As a result, cell structures are visualized directly, with image contrast being derived from differences in biochemical composition and density. For example, dense lipid-rich structures exhibit significantly greater X-ray absorption than organelles such as vacuoles that have significant water content.

  31. Schematic representation of the main components in XM-2, a new soft X-ray microscope designed for cellular imaging.

  32. Soft X-ray tomography • As with light and electron microscopes a soft X-ray microscope can only produce 2-D representations of a 3-D specimen. • However, if a sufficient number of 2-D images are recorded at incremental angles around a rotation or ‘tilt’ axis, a 3-D tomographic reconstruction of the specimen can be calculated.

  33. A 3-D reconstruction of two yeast cells in a glass capillary (top center) calculated from 2-D projections collected sequentially around a rotation axis (shown at 30° increments).

  34. Sample preparation • Biological materials are eventually damaged when they are exposed to intense sources of radiation (doesn’t matter whether UV, fluorescence, electrons, or X-ray) • Tomographic imaging requires a number of projection images to be collected from the same specimen. Thus, we must not alter/destroy the sample. • The most efficient way to prevent this is to mount the specimen in a cryogenic stage and image it after vitrification. • Vitrification = the transition of a substance into a glass. • In this context vitrification = the proper cooling of the samples in a manner that prevents ice crystal formation and structural changes/damages.

  35. Comparative images of the fission yeast Schizosaccharomyces pombe using light and soft X-rays. (a) Conventional fluorescence microscopy (vacuoles labelled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) from Invitrogen). (b) Differential interference contrast (DIC) light microscopy. (c) Orthoslices through a soft X-ray tomographic reconstruction. (d) The reconstruction in (c) after segmentation. Organelles are identified by their characteristic shape and measured X-ray linear absorption coefficients (LAC). Key: nucleus, blue; nucleolus, orange; mitochondria, grey; vacuoles, white; lipid-rich vesicles, green. The scale bar represents 1 μm. Proteins can be located by GFP-tags

  36. Surface plasmon resonance imaging for affinity-based biosensors.Scarano S, Mascini M, Turner AP, Minunni M.Biosens Bioelectron. 25(5):957-966(2010).

  37. a) Kretschmann configuration for SPRi; a high refractive index prism is in contact with the detection cell and couples the incident light to the surface plasmons by evanescent waves. p-polarised light is directed to the prism, on which the biomolecular probe is tethered, and a CCD camera collects the output signal as variations in reflectivity. (b) Data are recorded as intensity variation of the reflected light at a fixed angle for each ROI selected. A differential image (left) is produced in real time together with the relative sensorgrams. As example, two sets of signals are reported here corresponding to the interaction with different chemically modified areas. During the specific interaction with the target analyte, only the relatively specific probe will react, leading to a local change in intensity of reflected light. This translates into black/white contrast for the image. The unspecific receptor series (used as negative control) will give negligible or no signal. (c) Sensorgrams corresponding to the interactions of the analyte with the spots on the surface.

  38.  Immobilisation methods using chemical linkers. A self-assembled monolayer is created as the foundation of the array comprising alkanethiols containing ω-terminated amine, hydroxylic or carboxylic functional groups. After the formation of the thiolated layer many immobilisation chemistries can be performed.

  39. Immobilisation methods exploiting direct attachment of the probe to the gold surface of the chip using thiol- and pyrrole-functionalised biomolecules.

  40. Signal improvement in SPRi-based on mass/colorimetric approaches. The upper diagram shows the DNA hybridisation step followed by addition of streptavidin and biotinylated DNA for signal amplification. In the lower drawing, an aptamer array is exposed to the ligand protein (VEGF) and then biotinylated antibody forms a sandwich-type assay. The SPRi signal was amplified using an anti-biotin conjugated horseradish peroxidase (HRP) that in presence of a suitable substrate creates a localised dark-blue precipitation reaction.

  41. SPR imaging-based monitoring of caspase-3 activation.Park K, Ahn J, Yi SY, Kim M, Chung BH.Biochem Biophys Res Commun. 368(3):684-9 (2008).

  42. Schematic diagram of the SPR imaging system for the detection of caspase-3 activity. The purified GST:DEVD:EGFP reporter protein was immobilized onto a glutathionylated gold chip surface, and subsequently analyzed via SPR imaging in response to caspase-3. The caspase-3-dependent cleavage of DEVD results in the removal of EGFP from the GST:DEVD:EGFP attached onto the chip surface, thereby allowing for alterations in the SPR images. EGFP = enhanced green fluorescent protein ~25 kDa

  43. Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging.Mori T, Inamori K, Inoue Y, Han X, Yamanouchi G, Niidome T, Katayama Y.Anal Biochem.375(2):223-31 (2008).

  44. (A) Schematic representation of microarray fabrication. (B) Microarray pattern of peptides on SPR imaging chip. (C) SPR images of peptide microarrays reacted with c-Src and PKA. In panel B, each peptide was spotted in triplicate on the chip.

  45. Signal amplification • Anti-pTyr antibodies were used after treatment with Tyr kinases. • The commercially available pSer/pThr antibodies did not have sufficient affinity and selectivity !!! • Thus, after treatment with Ser/Thr kinases a phosphate-specific chelate compound modified with biotin (Phos-tag biotin) was used. The biotin was reacted with streptavidin and that was detected with the appropriate antibody.

  46. A cautionary tale: each method measures only what it was designed to detect Project: identifying binding site residues of heme-coordinating proteins by linking them to the porphyrin ring Oxidative shift Forms radical upon UV-activation

  47. Everything is hunky-dory UV spectrum of native Mb ( ), the Mb Fe−aryl complex (- - -), and the Mb Fe−aryl complex after photolysis (− −):  (A) complex formed with (meta-azidophenyl)diazene and (B) complex formed with (para-azidophenyl)diazene.

  48. What did mass spectrometry find ? • On the protein level – expected MW before the oxidative shift; multiple side chain oxidation detected after that; complete mess after UV-activation • On the peptide level – all kind of unexpected side-reactions; porphyrin-ring fragment-attachments; proton extraction i.e. double bond formation – all over the protein

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