Topic 2: Membrane Proteins

How do we observe protein movements in the membrane? 1. Sit back and grab a FRAP 2. Or don't and FLIP out 3. Cell Fusion Technique or CFT

FRAP: Fluorescent Recovery After Photobleaching - take membrane, bleach w/ laser beam (won't fluorece) , observe recovery time of fluorescene in bleached area FLIP: Fluorescene Loss In Photobleaching - take membrane, bleach w/ laser beam, have a separate measurement area (will fluorece) and bleached area (won't fluorsce) observe recovery time of fluorescene in bleached area Both techniques measure large (only) populations of protein covering large areas Thus, lack of movement of subpopulations or compartmentalization would not be detected CFT: Take two diff cell types, i.e. mouse and human membrane proteins, hybrid cell via virus-induced fusion, label with fluorescent antibodies, labeled proteins mix, witness both and diffusion of plasma membrane proteins w/ time.

Side Chains w/ Unique shit: "Grandma Craft Party"

Glycine, Gly, "G-randma": -non-polar -side chain had one single hydrogen atom, can fir into hyrdro-phillic & phobic side Cysteine, Cys, "C-raft" -Polar/uncharged -forms covalent bonds w/ other cysteine =disulfide link Proline, Pro, "P-arty" -non-polar -hyrdrophobic side chain, can get kinky disrupting 2ndary structure

Non-Polar side chains: "Grandma Always Visits London In May For Winston's Party"

Glycine, Gly, G Alanine, Ala, A Valine, Val, V Leucine, Leu, L Isoleucine, Ile, I Methionine, Met, M Phenylalanine, Phe, F Tryptophan, Trp, W Proline, Pro, P To remember that that this is the acronym for non-polar side chains, recall that London, England, is not located near the Earth's poles. Also, it may help you to associate London with Winston Churchill (the United Kingdom's prime minister during World War II) and, by extension, with May 8th (Victory in Europe Day, which celebrates the formal surrender of Nazi Germany to the Allies).

Transmembrane proteins & Aquaporins: Phobic Outsides and Philic insides

Aquaporins create a hydrophilic channel (inside) in the membrane, allowing water passage.

Polar/ Electrically-charged side chains: "Dragons Eat Knights Riding Horses"

Aspartate, Asp, D Glutamate, Glu, E Lysine, Lys, K Arginine, Arg, R Histidine, His, H By process of elimination, you'll know that this acronym refers to the remaining group of amino acids. Like those pesky dragons, nearly every molecule here is a troublemaker: aspartate (D), glutamate (E), lysine (K), and arginine (R).

The Cytosol; A Reducing Environment?

Reducing Environment: atmospheric condition in which oxidation (Oxidation is loss of electrons, i.e ) is prevented by removal of oxygen Example: during the combustion of wood, oxygen from the air (oxidant) is reduced, gaining electrons from the carbon (reductant). In redox processes, the reductant transfers electrons to the oxidant. It turns out that the reducing environment of the cytosol is mainly regulated by special reducing enzymes. These regulate the redox balance in the cytosol by keeping reactive oxygen species (ROS) at very low levels.

Membrane bending Broteins; Deforming our bilayers:

Required for many processes including vesicle budding, cell movement, and cell division. Causing curvature in the membrane. 3 Major Mechanisms: 1. insert into one leaflet via hydrophobic domains or lipid anchor. increases area of that leaflet causing bending. Found in ER tubules. 2. create rigid scaffold that stabilizes curved membrane. Found in coat proteins that create vesicles. 3. Lipid binding proteins that cause local increase in concentration of one type of lipid, inducing curvature.

Summary A Major component of the plasma membrane is the ___________ _______________ that attaches the membrane to the interior and limits movement of proteins and lipids.

cortical cytoskeleton

Summary What is a GPI-anchor protein again? Remember, they are a lipid Anchored Membrane protein.

glycophosphatidylinositol, or GPI in short, is a glycolipid that can be attached to the C-terminus of a protein during posttranslational modification. Proteins containing a GPI anchor play key roles in a wide variety of biological processes GPI-linked) proteins contain a signal sequence, thus directing them to the endoplasmic reticulum (ER). The protein is co-translationally inserted in the ER membrane via a translocon and is attached to the ER membrane by its hydrophobic C terminus; the majority of the protein extends into the ER lumen. The hydrophobic C-terminal sequence is then cleaved off and replaced by the GPI-anchor. As the protein processes through the secretory pathway, it is transferred via vesicles to the Golgi apparatus and finally to the plasma membrane where it remains attached to a leaflet of the cell membrane. Phospholipase C (PLC) is an enzyme that is known to cleave the phospho-glycerol bond found in GPI-anchored proteins. Treatment with PLC will cause release of GPI-linked proteins from the outer cell membrane.

Transmembrane proteins & rooming with bilayer.

in most transmembrane proteins, the polypeptide chain crosses the lipid bilayer in an alpha helical conformation -Amiphathic- both polar and non-polar domains (part that's inside is hydrophobic and the part outside is hydrophilic)

Summary Modifications of transmembrane proteins can be done by adding oligoaccharides to the protein: -occurs in the ER or Golgi so ends up on non-cytosolic side -Disulfides also occur on this side, prevented on cytosolic side to due reducing environment "What is the significance of these additions"

**[oligo= "a few"], [sacchar = "sugar"] oligosaccharide chains linked to lipids or to compatible amino acid side chains in proteins, by N- or O-glygosidic bonds. N-linked oligosaccharides are always pentasaccharides attached to asparagine via a beta linkage to the amine nitrogen of the side chain. Process of N-linked glycosylation occurs cotranslationally, or concurrently while the proteins is being translated. Since it is added cotranslationally, it is believed that N-linked glycosylation helps determine the folding of polypeptides due to the hydrophilic nature of sugars. Alternately, O-linked oligosaccharides are generally attached to threonine or serine on the alcohol group of the side chain. O-linked glycosylation occurs in the golgi apparatus, where monosaccharide units are added to a complete polypeptide chain. Cell surface proteins and extracellular proteins are O-glycosylated. Glycosylation sites in O-linked oligosaccharides are determined by the secondary and tertiary structures of the polypeptide, which dictate where glycosyltransferases will add sugar

Summary These proteins frequently span membranes w/ either ____________, that contain many hydrophobic residues on their surface or ________________ structures.

Alpha Helices Beta Barrels

All Alpha Helix and No Beta Barrels: b-barrels form transmembrane channels too!!!

-usually found in outer membrane of mitochondria , chloroplasts, and bacteria -can function as proteins that are: 1. virus receptor 2. lipase (hydrolize lipids) 3. porin 4. iron transporter

The 6 manners of movement (or lack thereof) for proteins in membranes. ** think 1. walking 2. anchors 3. motor 4. land locked 5. fences 6. ExCell traffic jams

1. moves freely (walking) 2. anchored below by membrane associated structures (anchors) 3. actively moved by motors attached to membrane associated structures (motor) 4. restricted by interactions w/ other integral membrane proteins (landlocked) 5. restricted by "a fence" created by underlying structures ( fence, duh) Cortical cytoskeleton 6. restricted by extracellular matrix (traffic jams)

Protein Structure & Function

-Polar residues tend to be on the surface of the protein -nonpolar residues tend to be pushed to the interior of the protein -sometimes, polar residues project into the hydrophobic regions giving the enzyme its catalytic activity

RBC cortical cytoskeleton & its role in constraining protein movement:

-cortical cytoskeleton & associated proteins form a lattice work on the surface of the plasma membrane and are fixed by their interactions w/ respective membrane domains -follow single molecules - people with RBC pathologies/ mutations in cortical cytoskeleton proteins show altered migration patterns for band 3

Peripheral Membrane Proteins and their Diverse Functions:

1. Form a protein skeleton on the inner membrane (cytoplasmic) surface 2. Provide mechanical support 3. Help to anchor integral membrane proteins 4. Some functions as enzymes 5. Transmit cellular signals

Lipid Anchored Membrane Protein

1. GPI - [glycosyl-phosphatidy-linositol anchored protein]- on external surface - removed & made soluble by detergents - penetrate hyrdophobic core of bilayer - requires & is inserted into bilayer by phospholipids Can Function as: -receptors -enzymes -cell adhesion -membrane structural proteins 2. Lipoproteins -proteins attached to cytoplasmic side of bilayerw/ long hydrocarbon chains/F.A or Prenyl groups - removed & made soluble by detergents - Does NOT penetrate the hyrdophobic core of bilayer - requires phospholipids - attached to bilayer through F.A. or preynl group Can Function as: -signaling molecules

Insane proteins & their movement in the membrane:

1. Some diffuse through the membrane randomly 2. some are immobilized in the membrane 3. some move only in specific directions 4. most move randomly through the membrane but w/ highly limited range 5. movement is thought to be restricted by interactions between [cytoplasmic domains of transmembrane proteins] & [structures in the cytoplasm] a & b\ (a) proteins anchored to the cytoskeleton (b) proteins anchored to the peripheral membrane proteins such as those that compromise the membrane skeleton 6. extracellular (ExCell) domains may also get entangled in ExCell structures, limiting mobility

The 4 ways that restrict proteins to membrane domains. **Think large groups of proteins hanging out on top, on bottom, and sides of cells.

1. assemble into larger aggregates 2. tethered by proteins inside the cell 3. inside the cell 4. interactions w/ proteins on other cells

What are the 3 Classes of Membrane Proteins?

1. integral protein: penetrate the bilayer aka transmembrane proteins. - removed & made soluble by detergents - penetrate hyrdophobic core of bilayer - inserted into bilayer - requires phospholipids 2. peripheal protein: associated w/ the outside of the bilayer via non-covalent bonds - removed by salts, high/low pH - h2o soluble - does not penetrate hydrohobic core of bilayer - associated at the surface of bilayer - no phospholipids required 3. lipid-anchored protein: outside the bilayer and anchored to lipid/glycolipid inside the bilayer - removed & made soluble by detergents - does not penetrate hydrohobic core of bilayer - attached to bilayer through F.A. or prenyl group - require phospholipids ** Monotopic proteins: technically a integral protein BUT there polypeptide chains do not cross the bilayer

What are the major proteins of RBCs'?

1. spectrin - resembles a double helix twizzler bridge thing on the surface of the plasma membrane - heterodimer - flexible rod - 25% of membrane protein mass - principle cortical cytoskeleton protein 2. band 3 - resembles a double barrel shotgun, holds ankyrin, which associates with the spanning spectrin rod on the surface of the plasma membrane - couple anion (negatively charged) HCO3- transporter - 12 multipass proteins - makes RBC permeable to HCO3- 3. glycophorin -resembles a poodle balloon animal, big balls (aka legs of the animal) on the inside of the plasma membrane and small balls (ears), on the surface of the plasma membrane - 1 of 2 major proteins on outer surface of RBC - 131 A.A, single pass homodimer (protein formed by two identical proteins) - glycolysated, w/100 sugar residues - fucntion: gives RBCs Hydrophilic charged coat that enables them to circulate w/o adhering to other cells - N-terminal on the outside of the lipid bilayer, longest chain, and C-terminal on the inside of the lipid bilayer

Summary Membrane _________ is important in many cellular processes and is regulated by proteins in several ways. Name those ways.

Curvature Required for many processes including vesicle budding, cell movement, and cell division

Summary ________________ can be used to solubilize ____________ membrane proteins for analysis. What technique would you use to identify transmembrane segments of those proteins?

Detergents, integral, Hydropathy plot Hydropathy plots allow for the visualization of hydrophobicity over the length of a peptide sequence. A hydropathy scale which is based on the hydrophobic and hydrophilic properties of the 20 amino acids is used. The larger the number on the index, the more hydrophobic the amino acid.

Modifications of transmembrane proteins: Ogliosacchaides & Disulfides

Ogliosacchaides are added to the protein, occurs in the ER and golgi and end up on the non-cytosolic side Disulfides also occur on this non-cytosolic side due to the reducing environment of the cytosol It turns out that the reducing environment of the cytosol is mainly regulated by special reducing enzymes. These regulate the redox balance in the cytosol by keeping reactive oxygen species (ROS) at very low levels. ... Other enzymes work by removing ROS and oxidation products from the cytosol

Polar/ UN-charged side chains: "Santa's Team Crafts New Quilts Yearly"

Serine, Ser, S Threonine, Thr, T Cysteine, Cys, C Asparagine, Asn, N Glutamine, Gln, Q Tyrosine, Tyr, Y To remember that this is the acronym for polar side chains, all you need to do is ask a five year-old where Santa and his team of elves live-at the North Pole, of course!

Insertion & Activation of multipass membrane proteins:

Single-pass membrane proteins cross the membrane only once, while multi-pass membrane proteins weave in and out, crossing several times. Single pass TM proteins can be categorized as Type I, which are positioned such that their carboxyl-[C] terminus is towards the cytosol, or Type II, which have their amino- [N] terminus towards the cytosol. -the same interactions that allow alpha helix to reside in the membrane (side chains and charges) drive formation of the final folded protein

Trans-membrane proteins, and their bilayer associations.

Transmembrane proteins can have: 1. single helix 2. several helix 3. beta barrel -exposed to only one side, & anchored by- 4. helix (monotopic) 5. covalently attached lipid 6. ogliosaccharide linker to lipid/ GPI anchor 7. non-covalent bond to proteins or lipids (either side)

Summary These proteins are associated with the membrane in one of three basic ways. a)I-P b)P-P c)L - P

a) Integral Proteins - penetrate the bilayer/ transmembrane protein b) Peripheral Proteins - associated w/ the surface of the membrane by noncovalent bonds (weak interactions) c) Lipid- anchored proteins - located outside the bilayer but anchored to a lipid / glycolipid w/n the bilayer

What are detergents?

are small amphiphatic molecules that are more H2O soluble than lipids can solubilize many membrane bound proteins allowing them to remain folded & studied Polar heads can be: -charged (ioninc) like SDS -Uncharged (non-ionic) like triton x Form Micelles above a certain Critical Concentration (CMC)

Why are there so many carbs on this Cell's surface?

carbohydrates found on lipids, tranmembrane proteins and peripheral membrane proteins regulate cell-cell interactions protect cells from mechanical and chemical damage contribute to the extracellular matrix

Bacteriorhodopsin

is a light driven proton (H+) pump that traverses the lipid bilayer as 7 alpha helices. A. retinal is contently attached to lysine side chain inside the structure. light causes the retinal to change shape allowing it to accept a proton from aspartic acid. this catalyzes a chain of transferase resulting in pumping a proton across the membrane B. a specific set of lipids associates with the complex holding it in place In this case the integral membrane protein spans the phospholipid bilayer seven times. The part of the protein that is embedded in the hydrophobic regions of the bilayer are alpha helical and composed of predominantly hydrophobic amino acids. The amino [N] terminal end of the protein is in the cytosol, while the C terminal region is in the outside of the cell. A membrane that contains this particular protein is able to function in photosynthesis

Summary Membranes contain __________ & ______________.

proteins and lipids