SYLICA Molecular Interactions – Bowater Feb 2013 SYLICA 2013 Bowater lectures Biophysical Methods to Study Molecular Interactions Bowater Lectures in Brno, Feb. 2013 4 lectures on linked topics will be delivered during the coming week: •Contemporary DNA Sequencing Technologies – 26/2/2013 @ 10:00 •Using ‘Omic Technologies to Investigate Gene Function – 26/2/2013 @ 14:00 •Biophysical Methods to Study Molecular Interactions – 27/2/2013 @ 10:00 •Synthetic Biology & Nanotechnology: Tomorrow’s Molecular Biology? – 28/2/2013 @ 10:00 SYLICA Molecular Interactions – Bowater Feb 2013 Molecular Interactions •For biological systems to function, interactions occur between many different types of molecules: DNA, RNA, Protein, Lipids, etc. •To ensure that biological systems function appropriately, such interactions must be carefully regulated •Wide range of Biophysical Chemistry approaches are useful for studying these interactions • SYLICA Molecular Interactions – Bowater Feb 2013 Bonds & Molecular Interactions ·Interactions between molecules are central to how cells detect and respond to signals and affect: ØGene expression (transcription & translation) ØDNA replication, repair and recombination ØSignalling ØAnd many other processes.... ·Interactions are (mainly) mediated by many weak chemical bonds (van der Waals forces, hydrogen bonds, hydrophobic interactions) ·Accumulation of many bonds influences affinity and specificity of interactions SYLICA Molecular Interactions – Bowater Feb 2013 Biophysical Chemistry Approaches for Studies of Molecular Interactions •Wide range of Biophysical Chemistry approaches are useful for studying molecular interactions: ØNMR ØX-ray crystallography ØSPR ØITC ØCD ØGel electrophoresis ØEPR ØMass spectrometry ØFluorescence • SYLICA Molecular Interactions – Bowater Feb 2013 In vitro In vitro and in vivo (?) Will also discuss other types of in vivo studies Biophysical Chemistry Approaches for Studies of Molecular Interactions •Wide range of Biophysical Chemistry approaches are useful for studying molecular interactions: ØNMR ØX-ray crystallography ØSPR ØITC ØCD ØGel electrophoresis ØEPR ØMass spectrometry ØFluorescence • SYLICA Molecular Interactions – Bowater Feb 2013 Many of these techniques are particularly useful for determining the strength (affinity) of interactions Protein-Nucleic Acid Interactions •A wide range of Biophysical Chemistry methods have been used to study interactions between proteins and nucleic acids •Particularly good for determining the strength (affinity) of the interactions ØHigh affinity, μM – nM: tend to involve sequence-specific interactions, e.g. restriction enzymes ØLow affinity, mM – μM: proteins tend to recognise aspects of “overall” structure i.e. not sequence-dependent SYLICA Molecular Interactions – Bowater Feb 2013 EMSA (“Gel Shift” Assay) •Electrophoretic Mobility Shift Assay (EMSA) or “gel shift” can provide information about protein-NA interactions SYLICA Molecular Interactions – Bowater Feb 2013 [Protein] DNA + protein DNA + protein + Ab M DNA alone A fairly straightforward technique, but only provides convincing data for high affinity interactions (typically <μM) “Footprinting” is a Technique to Identify a DNA-binding site •Premise: DNA bound by protein will be protected from chemical cleavage at its binding site 1)Isolate a DNA fragment thought to contain a binding site and “label” it 2)Bind protein to DNA in one tube; keep another as a “naked DNA” control 3)Treat both samples with chemical or enzymatic agent to cleave the DNA 4)Separate the fragments by gel electrophoresis and visualize bands on X-ray film or imager plate SYLICA Molecular Interactions – Bowater Feb 2013 Protein-DNA Footprinting SYLICA Molecular Interactions – Bowater Feb 2013 BOX 26-1 FIGURE 1 Footprint analysis of the RNA polymerase–binding site on a DNA fragment. Separate experiments are carried out in the presence (+) and absence (–) of the polymerase. Footprinting Results of RNA Polymerase Bound to Promoter SYLICA Molecular Interactions – Bowater Feb 2013 BOX 26-1 FIGURE 2 Footprinting results of RNA polymerase binding to the lac promoter (see Fig. 26–5). In this experiment, the 5’ end of the nontemplate strand was radioactively labeled. Lane C is a control in which the labeled DNA fragments were cleaved with a chemical reagent that produces a more uniform banding pattern. Binding of Proteins to DNA Often Involves Hydrogen Bonding •Gln/Asn can form specific H-bond with Adenine’s N-6 and H-7 H’s •Arg can form specific H-bonds with Cytosine-Guanine base pair SYLICA Molecular Interactions – Bowater Feb 2013 •Major groove is right size for a-helix and has exposed H-bonding groups DNA-binding domains •Proteins generally recognise aspects of nucleic acid sequence, or variations in structure and/or flexibility •High-resolution structures of many protein-DNA complexes have now been solved •Similar structural domains occur in different proteins: ØHelix-turn-helix ØZinc-finger ØZinc-binding domain ØBasic region-leucine zipper (bZIP) Øβ-sheet recognition SYLICA Molecular Interactions – Bowater Feb 2013 The Helix-turn-helix Motif is Common in DNA-binding Proteins •Each “helix-turn-helix” covers ~ 20 aa ØOne a-helix for DNA recognition, then b-turn, then another a-helix ØSequence-specific binding due to contacts between the recognition helix and the major groove SYLICA Molecular Interactions – Bowater Feb 2013 •Four DNA-binding helix-turn-helix motifs in the Lac repressor Helix-turn-helix ·Helix-turn-helix is most common observed DNA-binding unit in prokaryotes · · · · · · · · Berg, Tymoczko & Stryer, “Biochemistry”, 5th edn, 2002, p. 874 · Note that 34 Å corresponds to 1 turn of DNA SYLICA Molecular Interactions – Bowater Feb 2013 Zinc-finger ·One of best-studied examples of DNA binding domain, but also binds RNA ·Each covers ~30 aa ·Binding is relatively weak, so typically there are a series of zinc fingers SYLICA Molecular Interactions – Bowater Feb 2013 ·“Finger” portion is a peptide loop cross-linked by Zn2+, which is usually coordinated by 4 Cys, or 2 Cys + 2 His ·One type of consensus sequences is: “Cys2/His2”: Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His ·Zinc is held in tetrahedral structure by conserved Cys and His Zn++ Zn++ = Cys = His SYLICA Molecular Interactions – Bowater Feb 2013 ·Regulatory protein Zif268, complexed with DNA Zinc Finger Motif is Common in Eukaryotic Transcription Factors FIGURE 28–12 Zinc fingers. (PDB ID 1ZAA) Three zinc fingers (shades of red) of the regulatory protein Zif268, complexed with DNA (blue). Each Zn^2+ coordinates with two His and two Cys residues. β-recognition motif ·In some prokaryotic regulatory proteins, this is an alternative DNA-binding motif ·E. coli methionine repressor binds DNA through insertion of pair of β-strands into major groove · Berg, Tymoczko & Stryer, “Biochemistry”, 5th edn, 2002, p. 874 SYLICA Molecular Interactions – Bowater Feb 2013 Protein-protein Interactions ·Various techniques are used to investigate protein-protein interactions, including: ·Biochemical/biophysical ØIsothermal calorimetry ØSurface plasmon resonance (e.g. BIACore) ØMass spectrometry e.g. from protein complexes Ø“Pull-down” assays – one protein can be bound by an antibody (immunoprecipitation) or via a “tag” ·Molecular/cellular biological ØTwo-hybrid experiments ØFluorescent proteins SYLICA Molecular Interactions – Bowater Feb 2013 Identifying Protein–Protein Interactions •Protein complex isolation ØEpitope tag one protein in the complex ØGentle isolation of epitope-tagged protein will also isolate stably interacting proteins ØAll proteins isolated can be separated and identified • SYLICA Molecular Interactions – Bowater Feb 2013 SYLICA Molecular Interactions – Bowater Feb 2013 •Use of Tandem Affinity Purification (TAP) tags has enhanced the procedure •Allows two purification steps eliminating loosely associated proteins, and minimizing non-specific binding Procedure for TAP–Tagged Proteins FIGURE 9–20 Tandem affinity purification (TAP) tags. A TAP-tagged protein and associated proteins are isolated by two consecutive affinity purifications, as described in the text. SYLICA Molecular Interactions – Bowater Feb 2013 •Protein of interest tagged with the GAL4-activation domain •DNA library with all yeast genes tagged with Gal4-binding domain •Reporter gene under the control of Gal4 •Differentially tagged proteins must interact in order to get expression of the reporter gene • Yeast-Two Hybrid System Similar techniques developed to use with bacterial and mammalian cells FIGURE 9–21 Yeast two-hybrid analysis. (a) The goal is to bring together the DNA-binding domain and the activation domain of the yeast Gal4 protein (Gal4p) through the interaction of two proteins, X and Y, to which one or other of the domains is fused. This interaction is accompanied by the expression of a reporter gene. (b) The two gene fusions are created in separate yeast strains, which are then mated. The mated mixture is plated on a medium on which the yeast cannot survive unless the reporter gene is expressed. Thus, all surviving colonies have interacting fusion proteins. Sequencing of the fusion proteins in the survivors reveals which proteins are interacting. Assessment of Protein-protein Interaction Data ·Currently believed that yeast has >30,000 different interactions (for ~6,000 proteins) ·Variety of studies using yeast (see von Mering et al. (2002) Nature, 417, 399-403) ·Overall conclusion is: different techniques identify different complexes! ·Results from protein-protein interaction studies should be confirmed by more than one experimental technique ·Especially important for considering if in vitro observations are relevant for in vivo situations · SYLICA Molecular Interactions – Bowater Feb 2013 Study of Protein-protein Interactions In Vivo ·Popular technique is “Two-hybrid” screen (yeast, mammalian or bacterial) ·Various fluorescent techniques are also in use: ØFRET – fluorescence resonance energy transfer; reports on distance between 2 fluorophores ØFluorescent reporters – expressed proteins emit fluorescence at specific wavelength ØFRAP (FLIP) – fluorescence recovery after photobleaching (fluorescence loss in photobleaching); allow movement of reporters to be monitored · SYLICA Molecular Interactions – Bowater Feb 2013 Fluorescence can be used to Determine Protein Location In Vivo •Use recombinant DNA technologies to attach Fluorescent Proteins to protein of interest ØVisualize with a fluorescent microscope •Immunofluorescence ØTag protein with primary antibody and detect with secondary antibody containing fluorescent tag ØProtein can also be fused to a short epitope and the primary antibody detecting the epitope can be fluorescently labeled SYLICA Molecular Interactions – Bowater Feb 2013 Fluorescently-tagged Proteins •Combination of molecular and cell biological studies analyse in vivo localisation of proteins expressed with a fluorescent “tag” •Important that “tag” does not interfere with protein activity • • • • Bastiaens & Pepperkok (2000) TiBS, 25, 631-637 •Can examine localisation of proteins containing different fluorophores SYLICA Molecular Interactions – Bowater Feb 2013 Green Fluorescent Protein Tags •Widely used tag is “Green fluorescent protein” (GFP) •GFP was first discovered as a companion protein to aequorin, the chemiluminescent protein from Aequoria victoria © C. Mills, Univ. Wash. SYLICA Molecular Interactions – Bowater Feb 2013 Green Fluorescent Protein Tags ·For GFP, the chromophore is a p-hydroxybenzylidene-imidazolidone (green background) ·Consists of residues 65-67 (Ser - dehydroTyr - Gly) of protein and their cyclized backbone forms the imidazolidone ring ·Peptide backbone is shown in red · · · · SYLICA Molecular Interactions – Bowater Feb 2013 Green Fluorescent Protein Tags ·Amino acid sequence SYG can be found in a number of other non-fluorescent proteins, but it is usually not cyclized, and Tyr is not oxidized ·Implies that this tripeptide does not have intrinsic tendency to form such a chromophore SYLICA Molecular Interactions – Bowater Feb 2013 GFP chromophore Development of Fluorescent Tags •Mutagenesis studies yielded GFP variants with improved folding and expression properties •Changes help: Øaccelerate speed and intensity of fluorophore formation Øhelp the molecule fold correctly at 37 °C Øovercome dimerization Øimprove expression by converting codons to those used by the organisms of interest •These characteristics are combined in the GFP variant known as enhanced GFP (EGFP) SYLICA Molecular Interactions – Bowater Feb 2013 GFP–Tagged Protein Localization SYLICA Molecular Interactions – Bowater Feb 2013 FIGURE 9–16 Green fluorescent protein (GFP). (a) The GFP protein (PDB ID 1GFL), derived from the jellyfish Aequorea victoria, has a β-barrel structure; the fluorophore (shown as a space-filling model) is in the center of the barrel. (b) Variants of GFP are now available in almost any color of the visible spectrum. (c) A GLR1-GFP fusion protein fluoresces bright green in Caenorhabditis elegans, a nematode worm (left). GLR1 is a glutamate receptor of nervous tissue. (Autofluorescing fat droplets are false colored in magenta.) The membranes of E. coli cells (right) are stained with a red fluorescent dye. The cells are expressing a protein that binds to a resident plasmid, fused to GFP. The green spots indicate the locations of plasmids. Further Development of Tags ·Continued efforts to engineer (or isolate) new fluorophores and reporter classes: Øbrighter and more red-shifted proteins useful for multi-spectral imaging and FRET-based methods Øincreased brightness will help track single molecules Ømore pH resistance useful in acidic environments ·Advances in imaging systems are also important: Ømore sensitive and quicker camera systems Øfilter systems for detecting different fluorophores Øsoftware for discriminating fluorescent signals ·Understanding complex protein interactions and dynamics also requires kinetic modeling and analysis Lippincott-Schwartz & Patterson (2003) Science, 300, 87-91 SYLICA Molecular Interactions – Bowater Feb 2013 GFP Turnover ·Analysis of protein turnover or temporal expression pattern and behavior is difficult with conventional GFP because the GFP chimeras are continuously being synthesized, folded, and degraded within cells ·Thus, at any particular time, proteins at different stages of their lifetime are being observed ·Several promising approaches have used FPs which have different fluorescent properties over time ·Another promising approach to studying protein lifetimes and turnover rates is the use of photoactivable fluorescent proteins Lippincott-Schwartz & Patterson (2003) Science, 300, 87-91 SYLICA Molecular Interactions – Bowater Feb 2013 GFPs in Action! ·Photoactivatable fluorescent proteins display little initial fluorescence under excitation at imaging wavelength (λ) ·Fluorescence increases after irradiation at a different λ – highlighting distinct pools of molecules within the cell ·Since only photoactivated molecules exhibit noticeable fluorescence, their behaviour can be studied independently of other newly synthesized proteins Lippincott-Schwartz & Patterson (2003) Science, 300, 87-91 SYLICA Molecular Interactions – Bowater Feb 2013 Immunofluorescence SYLICA Molecular Interactions – Bowater Feb 2013 FIGURE 9–17 Indirect immunofluorescence. (a) The protein of interest is bound to a primary antibody, and a secondary antibody is added; this second antibody, with one or more attached fluorescent groups, binds to the first. Multiple secondary antibodies can bind the primary antibody, amplifying the signal. If the protein of interest is in the interior of the cell, the cell is fixed and permeabilized, and the two antibodies are added in succession. (b) The end result is an image in which bright spots indicate the location of the protein or proteins of interest in the cell. The images show a nucleus from a human fibroblast, successively stained with antibodies and fluorescent labels for DNA polymerase , for PCNA, an important polymerase accessory protein, and for bromo-deoxyuridine (BrdU), a nucleotide analog. The BrdU, added as a brief pulse, identifies regions undergoing active DNA replication. The patterns of staining show that DNA polymerase and PCNA co-localize to regions of active DNA synthesis. One such region is visible in the white box. Identifying Regions Involved in Protein-protein Interactions ·Once protein-protein interactions have been identified, it is important to establish how the interactions occur e.g. what regions or specific amino acids are important for the interaction? ·Well-used approach is to prepare different fragments or mutations of proteins and see if there is any effect on the protein-protein interaction ·Results usually confirmed by more than one experimental technique SYLICA Molecular Interactions – Bowater Feb 2013 SYLICA Molecular Interactions – Bowater Feb 2013 •Protein of interest tagged with the GAL4-activation domain •DNA library with all yeast genes tagged with Gal4-binding domain •Reporter gene under the control of Gal4 •Differentially tagged proteins must interact in order to get expression of the reporter gene • Yeast-Two Hybrid System FIGURE 9–21 Yeast two-hybrid analysis. (a) The goal is to bring together the DNA-binding domain and the activation domain of the yeast Gal4 protein (Gal4p) through the interaction of two proteins, X and Y, to which one or other of the domains is fused. This interaction is accompanied by the expression of a reporter gene. (b) The two gene fusions are created in separate yeast strains, which are then mated. The mated mixture is plated on a medium on which the yeast cannot survive unless the reporter gene is expressed. Thus, all surviving colonies have interacting fusion proteins. Sequencing of the fusion proteins in the survivors reveals which proteins are interacting. Transient Protein-protein Interactions ·Current proteomics studies have allowed the identification of protein interactions on large scale ·Protein networks underline the multi-specificity and dynamics of complexes involving transient interactions Nooren & Thornton (2003) EMBO J., 22, 3486-3492 SYLICA Molecular Interactions – Bowater Feb 2013 ·Biophysical methods are very useful to characterise such interactions Molecular Interactions Overview •Biophysical chemistry approaches are good for studies of macromolecular interactions, particularly because they can provide quantitative data •High-resolution structures have been identified for a wide range of interactions; particularly well-defined for some proteins binding to nucleic acids •Many techniques developed to study protein-protein interactions in vivo •Applications of fluorescence and fluorescent proteins provide important information about macromolecular interactions SYLICA Molecular Interactions – Bowater Feb 2013