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Membrane Proteins.docx

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Western University
Biology 2382B
Jessica Kelly

Lecture 14 – Membrane Proteins Since pure phospholipid membranes are only semipermeable to a few small molecules and gases…and also function only to form a closed compartment…many biological functions of membranes are carried out by membrane embedded/associated proteins… Three Types of Membrane Proteins: 1. Integral 2. Lipid-linked 3. Peripheral All are asymmetric... (i.e. not found in equal abundance on both sides but rather localized to one leaflet or the other…) Water is on outside. Inside is hydrophobic, outside is hydrophilic 1. Integral Membrane Proteins Asymmetric  specifically orientated Three Distinct “Domains”:  Cytoplasmic (hydrophilic)  Transmembrane (hydrophobic)  Exoplasmic (hydrophilic) Transmembrane Domain: Hydrophobic secondary or tertiary structures that span the lipid bilayer…  Commonly α helices (approx. 20-25 amino acids long)  Arg and Lys (charged AAs) near cytosolic side  (+)ive charge cannot cross membrane of ER during synthesis = hydrophilic (interact w/ polar head groups)  β barrels (another transmembrane structure composed of layered beta sheets) Mostly glycosylated in exoplasmic domain… (Provides specificity for extracellular interactions)… 1 2. Lipid Linked Proteins These proteins are anchored to the membrane by lipophilic adduct… a) Acylation of Gly residue of protein @ N-terminus – (palimate or other fatty acid) b) Prenylation of Cys residue of protein @ C-terminus – (5C isoprene units) c) Glycosylphosphatidylinositol (GPI) anchor – PI lipid is covalently linked to sugar residues which are linked to phosphoethanolamine…this acts an anchor for a protein and is exclusively exoplasmic • Lipid linked proteins do not enter bilayer… • Lipid linked proteins (& most membrane proteins) have lateral mobility within the membrane… 3. Peripheral (non-covalent weak interaction) Proteins “Attached” through non-covalent interactions (relatively weak interactions)…  Ionic Interactions  Hydrogen Bonds  Protein-protein Interactions  Van der Waals forces Can act as adapter proteins and allow cytoskeletal filaments to associate with bilayer… (i.e. dystrophin & ankyrin that link integral membrane proteins to cytoskeleton) ** see diagram at beginning** 2 Insertion of Proteins into Membranes Topogenic Sequences – internal signal sequences of amino acids in a nascent peptide that direct its insertion into the target membrane/organelle by directing the translating ribosome to a translocon on the target membrane…they are recognized by their topology (i.e. charge & shape) & vary from protein to protein Three Types of Topogenic Sequences: 1. N-terminal signal sequence – signal sequence at the N-terminus that is cleaved upon entry into the ER lumen 2. Stop-Transfer Anchor Sequence (STA) – uncleaved & internal signal sequence composed of hydrophobic amino acid that becomes the membrane spanning - helix 3. Signal-Anchor Sequence (SA) – uncleaved & internal signal sequence composed of hydrophobic amino acid that becomes the membrane spanning - helix  (whether C or N terminus is in cytosol determined by location of (+)ive charged AA’s relative to STA) STA sequences stop the transfer (i.e. stop translation) into the lumen of the peptide as well as form an -helix transmembrane domain… **ALL proteins begin being translated in the cytosol & require a signal in order to transfer the protein to another organelle/organelle membrane…** 3 Synthesis of Type I Proteins Main Features: 1. N-terminal signal sequence (cleaved) 2. Stop-transfer anchor sequence (uncleaved/internal) • N-term signal sequence begins being translated in the cytosol… recognized by SRP which transfers the translating nascent protein-ribosome complex to the translocon on the ER membrane  Translocon = channel that allows the protein to be moved into the ER lumen • Type I transmembrane proteins  have their N-terminal sequence in the lumen and C-terminus in cytosol • STA sequence (specific shape and charge i.e. hydrophobic)  stops transfer of protein into the ER and transfers it to become the integral membrane spanning sequence the rest of the protein is translated in the cytosol N-terminus is in the lumen (extracellular) with the C-terminus in the cytosol as well as the STA sequence as the membrane spanning anchor region in the full synthesized type I transmembrane protein… 4 Synthesis of Type II and Type III Main Features: 1. Have signal-anchor sequence (internal). 2. Orientation determined by (+)ively charged amino acids (kept in cytosol) • No n-terminal sequences rather internal signal anchor sequence (SA) that becomes the membrane spanning domain Type III  SA sequence which is internal tells the SRP to transfer protein to ER N terminal domain is short and uncharged = lumen/exoplasmic… C-terminal domain is long and charged = cytosol Type II  SA sequence is internal, again signals SRP to transfer protein to ER but N-terminal domain is long and charged = cytosol…the C-terminal domain is short and uncharged = lumen/exoplasmic The orientation of (+)ively charged amino acids relative to the SA sequence will determine the orientation of the polypeptide in the membrane…these charged sequences always remain in the cytosol  orientation determines the difference between type 2 & 3 membrane proteins… 5 Synthesis of Type IV Proteins • Orientation of initial helix determined by (+)ively charged amino acids next to signal-anchor sequence (SA) • Have alternating signal-anchor sequences and stop transfer sequences • Can have even or odd number of transmembrane domains  can be oriented with N & C termini on the same side or opposite sides of the membrane Type IV A – multi-membrane spanning domains with both C & N termini on cytosol or lumen (even) Type IV B - multi-membrane spanning domains with C & N termini on opposing sides cytosol and lumen (odd) 6 Transport Across Membranes Cells must import & export large and/or charged molecules in order to sustain themselves  these cannot diffuse across the hydrophobic plasma membrane... 3 Major Types of Transport 1. Passive Diffusion 2. Facilitated Transport (i.e. pores, channels, gates, & uniporters…) 3. Active Transport (i.e. ATP pumps, symporters, & antiporters Passive Diffusion Partition coefficient (K) - a measure of the preference of a molecule to partition into a hydrophobic environment Higher K = more lipid soluble K = c /c aq Lower K = less lipid soluble The main determinants of diffusion are the partition coefficient and the concentration difference…  A molecule with a High K will move down its concentration gradient through a membrane…(high K  lipid soluble so not unfavourable to move through membrane) 7 Facilitated Transport Driving force  concentration gradient (moving down it is favourable i.e. – ΔG)… Types  pores, channels, gates, uniporters Movement of hydrophilic substances through a protein-lined pathway so they don’t come into contact with hydrophobic interior of membrane… • 1. Faster than predicted by passive diffusion (important helps speed up movement of solutes  diffusion alone too slow for cells) • 2. Specific  i.e. K+ channels will not allow Na+ ions through only K+ ions due to interactions of the ion with the protein not being favourable for Na+ ions • 3. Saturable  only so many on a membrane…at a certain concentration of solute all channels, gates, pores, etc… will be working at the fastest rate possible Facilitated Transport – Pores & Channels Integral membrane proteins create “hydrophilic holes” in the membrane that are large enough for solutes to pass through…ions move down gradient = no energy required… • Pores/Channels still have selectivity based on interactions with molecules passing through (i.e. K+ channels do not allow the smaller sodium Na+ ions to pass through them) How? • Na+ cannot form the favourable interactions with the protein channel (oxygen’s) that it forms with water based on its size (smaller then K+) 8 • In order to move through channel it must break it’s 8 interactions with H2O (hydration shell) and form new ones with channe
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