Lecture 5-Protein Structure Topology (March 8) PDF

Module 2: Protein Structure Topology BIOC*4580 (Winter 2024) Quiz 3 – Next Friday, March 15 Module 1 and 2 45 min – multiple choice – Short answer questions – Matching – Multiple Select 25% of the overall grade Determining Membrane Protein Structure ~20 - 30 % of the proteins in a given organism’s g...

Module 2: Protein Structure Topology BIOC*4580 (Winter 2024) Quiz 3 – Next Friday, March 15 Module 1 and 2 45 min – multiple choice – Short answer questions – Matching – Multiple Select 25% of the overall grade Determining Membrane Protein Structure ~20 - 30 % of the proteins in a given organism’s genome are membrane embedded However, less than 2% of structures found in PDB are of membrane proteins. The technical challenges in solving membrane protein structures makes this one of the most poorly understood protein groups Techniques for structure determination of membrane proteins X-ray crystallography (i) purification (ii) reconstitution (ii) crystallization Cryo electron microscopy – 2D crystals – Single particle AFM (2D view) Computational Methods (e.g. AlphaFold) Fluid girdlecannot mediate crystal contacts Large-scale protein purification is essential for studies of any protein need >100 mg of pure protein for crystallization trials for soluble proteins, expression of poly-His-tagged protein in E. coli has been successful for obtaining large amounts. BUT overproduction of integral proteins in E. coli is often lethal; special bacterial strains or other cell types (yeast, insect cells) are needed expressed proteins may form particles called inclusion bodies, which must be solubilized under denaturing conditions, and then the proteins re-folded Available membrane space limits expression Express soluble protein Express membrane protein Membrane Protein Topology – Bioinformatics & Molecular Biology Bioinformatics approaches are a first tool to help understand a membrane protein’s topology Such predictions can be used to guide molecular and cell biological experiments to probe portions of the protein that are within the membrane, or exposed on the cytoplasmic or extracellular surfaces Nomenclature of transmembrane proteins is based on TOPOLOGY 1.BITOPIC (not MONOTOPIC) ▪ single transmembrane (TM) segment, or insertion into membrane but not transmembrane per se ▪ only 5-10% of protein mass within bilayer (takes ~5 kDa to span), e.g., human erythrocyte glycophorin ▪ N-terminus extracellular (Type I) or intracellular (Type II) Nomenclature of transmembrane proteins is based on TOPOLOGY 2. POLYTOPIC (Type III) ▪ 2 or more transmembrane segments, e.g., bacteriorhodopsin (7TM) ▪ larger fraction of protein mass is embedded in bilayer (>60%) ▪ membrane-spanning segments are separated by β-turns, or longer loops, at membrane surface ▪ N- and C-termini can be either cytoplasmic or extracellular (all combinations possible) ▪ if even number of TM segments, N- and C-termini must be on the same side of the membrane ▪ if an odd number of TM segments, N- and C-termini must be on opposite sides of the membrane Bioinformatics of Integral Membrane Proteins Predicting Membrane Protein Topology from the Sequence Where are the termini, TM segments, β-turns, & loops? Common eukaryotic TM structural motif is α-helix. B&T 12.1 Hydrophobicity Analysis Every amino acid has a certain “hydrophobicity” quantity, expressed as a “hydropathy index” associated with it, based on: I. Experimental data – Free energy change associated with moving an amino acid side chain from a hydrophobic environment to water (GTr ) II. Statistical data – look in structural databases and see relative distribution of amino acids in protein exterior vs. its interior III. Aggregate scales – average together a selection of known scales to try and minimize systematic errors e.g., Kyte-Doolittle: most commonly used scale, good to ~1 decimal point Lehninger 8th ed. Hydrophobicity Analysis – the Kyte-Doolittle & Engelman-Steitz-Goldman Scales B&T 12.1 The values reflect the GTr ( Free energy of transfer) of an amino acid side chain from a hydrophobic environment into water. This value is favourable G 0; +ve for nonpolar side chains. Hydropathy plots To scan for membrane spanning sections of a protein, we determine the average hydropathy index for successive segments of a polypeptide chain by summing and averaging the hydropathy index of each amino acid in that section. Each segment is known as a “window” and is chosen to be between 7-20 amino acids in length. When calculating for a window of 7, the hydropathy index of amino acids 1 to 7, 2 to 8, 3 to 9 etc. is averaged. This technique is known as a “sliding window”. 1 n etc. Plotting each amino acid’s hydrophobicity individually leads to a very jagged plot Hydrophobicity Plots Sequence of your protein: aa1 aa2 aa3 … aai … aan-1 aan Hydrophobicity profile of protein (using the scale that you have chosen): h1 h2 h3 … hj … hn-1 hn “Sliding window” can be applied to sequence to average the local hydrophobicity: e.g. to derive the sliding window average of residue 10 with a window of 7: take the hydrophobicity values of residues 7 (n-3) to 13 (n+3) inclusive, and average them 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 Hydropathy plots The average hydropathy index for each segment is then plotted against the middle residue in each window (residue 4 for window 1-7) A region with more than 20 residues of high hydropathy index is indicative of a transmembrane segment. Glycophorin has a single transmembrane α-helix Bacteriorhodopsin has 7 transmembrane α-helices Hydrophobic transmembrane segments (e.g., α-helices) have a positive free energy for going aqueous Generally, a TM helix is predicted if the mean hydrophobicity (Kyte Doolittle) exceeds 1.4 The thermodynamics is compelling for the formation of transmembrane α-helices All amino acids can form 2 hydrogen bonds from backbone The non-polar domain of the membrane has a low dielectric constant. The per residue cost of disrupting H-bonds in the membrane is ~4 kcal/mol. For a 20-residue transmembrane segment, the free energy driving the formation of an α-helix is 80 kcal/mol. –NH–C=O– (peptide bond) ΔGtr from water to alkane Non-H-bonded +6.4 kcal/mol H-bonded +2.1 kcal/mol Dielectric constant=measures the ability of a substance to insulate charges from one another. It is low for non-polar and high for polar substances Luckey “Membrane Structural Biology” Non-standard helices complicate interpretation of hydropathy plots Half helices which partially span the membrane (e.g. K+ channel, aquaporin) generally won’t show up on hydropathy plots TM Helices can have significant numbers of hydrophilic residues facing into a ligand binding site or channel Helices that cross the membrane at a shallow angle may be unusually long TIBS 31, 106-113 (2006)