Lecture 6-Protein structure topology (March 11).pdf

Lecture 6 W24 Quiz 3 –Friday, March 15 Module 1 and 2 45 min – multiple choice – Short answer questions – Matching – Multiple Select 25% of the overall grade Hydropathy profiles of OMPs Stryer 5 Fig. 12.28 & 12.20 β-barrel OMPs have hydrophilic residues within the pore OMPs lack the clearly hydropho...

Lecture 6 W24 Quiz 3 –Friday, March 15 Module 1 and 2 45 min – multiple choice – Short answer questions – Matching – Multiple Select 25% of the overall grade Hydropathy profiles of OMPs Stryer 5 Fig. 12.28 & 12.20 β-barrel OMPs have hydrophilic residues within the pore OMPs lack the clearly hydrophobic regions of helical membrane proteins Simple hydropathy analysis fails to detect these proteins Computational recognition of OMPs OMPs are harder to recognize computationally than α-helical transmembrane proteins The alternating hydrophobic/hydrophilic residues as residues from the β-strand project alternately outwards and inwards is not strict as inner barrel residues are fairly frequently hydrophobic (e.g. where loops inserted into the barrel contact them). The fact that the β-strands are shorter than transmembrane helices (as few as 6 8 residues) means that there is much less signal available to detect individual membrane crossing elements. Computational recognition of OMPs cont. Homology to known b-barrel proteins is an obvious prediction tool Otherwise, OMP predictions rely on sequence features which predict the consensus properties of OMPs: – even numbers of b-strands – short periplasmic loops – N- and C- termini periplasmic – antiparallel strands – alternating aliphatic residues facing the membrane – Enrichment in aromatics Molecular Biology on the Interweb If you have a protein sequence, you can learn a lot about it by the tools available at, for example, the website: http://www.expasy.ch/proteomics – Generate Hydropathy Plots – BLASTp searches and alignments to see conserved residues – calculations of amphipathicity The result should be a testable topology model Exercise http://web.expasy.org/protscale Find the protein sequence from PDB or from Expasy-UnitProtKB Or Use UniProtKB ID for the protein – e.g. P02945 for bacteriorhodopsin; P13945 for human betaadrenergic receptor Can generate a hydropathy plot Biochemistry and Molecular Biology of Membrane Proteins Probing the TOPOLOGY of membrane proteins by experimental means Prokaryotes: – Gene fusion with a reporter gene, e.g., the gene encoding alkaline phosphatase (phoA fusions) Eukaryotes: – Epitope insertion & antibody labeling – Insertion of glycosylation sites – Cysteine mutagenesis and chemical modification with fluorescent or other probes The E. coli phoA gene is a useful “reporter” gene, especially for bacterial proteins Alkaline phosphatase (AP) encoded by phoA gene in E. coli. Wild-type phoA gene has a signal-sequence allowing export of AP into the periplasm where it is active. AP is NOT active in cytoplasm (highly reducing). Alkaline phosphatase activity can be detected on solid media containing X-P (a colourless compound cleaved to form a blue coloured compound like X-gal). “X” = a substituted indole here. The phoA-fusion strategy is straightforward but tedious 5' end of the gene of the protein of interest (encoding N-terminal end of the protein) is spliced to a gene for a "reporter" protein, or vice versa (e.g. PhoA) make many phoA fusions (15-25) at different points along the membrane protein gene thought to be within either intracellular or extracellular loops/turns Gene fusions to phoA provide an assay for extracytoplasmic proteins or domains of proteins. 3´ 3´ 5´ 5´ PhoA Protein phoA Gene Fusions use AP activity as “reporter” If phoA is fused to a domain of a protein that is in the periplasm, it will result in alkaline phosphatase activity (yielding an X-P+ colony). If phoA is fused to a domain of a protein that is in the cytoplasm, it will NOT result in alkaline phosphatase activity (yielding an X-Pcolony). High AP activity – blue; Medium – hatched green; Low – light blue arrows. + - Luckey “Membrane Structural Biology” phoA fusions to the lacY protein, lactose permease, (J Calamia & C Manoil, Proc. Natl. Acad. Sci. USA 87: 4937-4941, 1990). There are limitations of phoA The phoA fusion works well for bacterial proteins, and for eukaryotic proteins that can be expressed in E. coli. Only partial proteins (not full-length) are being expressed absence of the C-terminal regions of the protein may alter the way the rest of the protein folds or inserts into the membrane. For most eukaryotic proteins, other strategies are required. Probing an epitope by fluorescent antibodies or enzymatically (mainly eukaryotic) If you don’t have antibodies directed against an epitope already within the protein, then insert an epitope into the protein (into the extra and intra cellular loops) against which you do have antibodies Question: Is your epitope (small region of the protein, 6-8 aa's) on one side or the other of the cell membrane? Czachorowski et al. Transmembrane topology of the mammalian Slc11a2 iron transporter. Biochemistry 48, 8422-8434, 2009. Epitope Tagging (Czachorowski et al. Paper) Posted on Courselink: 1. Czachorowski, M., Lam-Yuk-Tseung, S., Cellier, M. and Gros, P. Transmembrane Topology of the Mammalian Slc11a2 Iron Transporter (2009). Biochemistry 48(35): 8422-8434 2. Associated work sheet Read the paper and find answers to the questions on the worksheet. You can leave out the “sequence analyses” and the “homology modelling” sections of the paper (methods, results and discussion). The work sheet should guide you through the sections that are relevant to our discussions. (Use for self study. You do not have to submit the work sheet to Courselink Dropbox.) Identify the location of the tag (and therefore the loop) using fluorescent antibodies against the epitope or enzymatically, under intact and permeabilized conditions Epitope detected in both intact and permeabilized cells – Extracellular Epitope detected only in permeabilized cells - intracellular Czachorowski et al. 2009 Summary – Antibody Labelling Requires Ab that binds to a well-defined epitope on the protein "site-directed antibodies“ can be generated by immunization of rabbits with a synthetic peptide corresponding to the selected region of protein a fluorescently-labelled Ab is used, and the cells are observed using a fluorescence microscope: immunofluorescence This technique can be used to probe receptor sites Insertion of a glycosylation site: an Asn-X-Ser/Thr motif An alternate to epitope insertion Glycosylation occurs only on EXTRACELLULAR loops Asn−X−Ser/Thr is (usually) N-glycosylated if present within an extracellular loop If intracellular, it is not accessible to glycosyl transferases and is therefore ignored Glycosylation sites are introduced (one at a time) into candidate loops by mutagenesis Recombinant proteins are expressed in cell culture, and tested for N-glycosylatation Glycosylated proteins run more slowly on SDSPAGE & treatment with N-glycanase alters their Mr If the protein has been glycosylated at the inserted site, N-glycanase treatment leads to a reduction in its Mr on SDS-PAGE The presence of glycosylation implies that the loop is extracellular # of N-linked Glycans After full treatment with N-glycanase The EMBO Journal (2003) 22, 1036 - 1046 N-glycanase- cleaves asparaginelinked oligosaccharides from glycoproteins. Insertion of Glycosylation Sites Need to engineer out any native glycosylation sites first Caution! inserting “unnatural” glycosylation sites does not guarantee that they will be glycosylated They may not be recognized properly by the glycosylation machinery, giving false negatives e.g. N-glycosylation does not occur if the site is too close to the membrane – need about 10 a.a. spacer. Cysteine mutagenesis – another alternative Cysteinyl residues have free thiol groups and are highly reactive, and can be inserted anywhere into a protein by site-directed mutagenesis, and then labeled at a specific site by a maleimide derivative May need to knock out endogenous cysteines, then insert cysteines by site-specific mutagenesis Will react with a free thiol here. Summary – Cysteine Mutagenesis 1 a Cys-less construct of the protein is made by replacing all the naturally existing Cys residues by Ala (conservative) (it has been found that such constructs are usually still functional) Cys residues are then introduced one at a time into specific loops/turns within the Cys-less protein, using site-directed mutagenesis of the existing amino acid Cys residues react covalently with various thiol-specific reagents, such as maleimide derivatives both membrane-permeant and membrane-impermeant maleimides are available, with tags to allow detection Summary – Cysteine Mutagenesis 2 each protein construct is expressed in intact cells, reacted with a permeant and an impermeant maleimide, and its location inferred permeant impermeant Extracellular Cys + + Intracellular Cys + - caution! Cys may be unreactive for other reasons, e.g., if located in a tightly folded region of the protein and inaccessible to reagent Complete Cysteine-scanning Mutagenesis and Site-directed Chemical Modification of the Tn10-encoded MetalTetracycline/H+ Antiporter The Journal of Biological Chemistry 276, 20330-20339, 2001. Caution with all Methods! modifications (epitope insertion, glycosylation site insertion, Cys mutagenesis) may have the following effects on a protein: – failure to target to plasma membrane correctly – incorrect folding leading to rapid degradation – altered folding leading to loss of function often, a large fraction of the constructs are unusable for one of these reasons Quiz 3 will test content up to here