Membrane Proteins

Describe fibrous proteins.

Highly elongated proteins whose secondary structures are the dominant structural motif. Found in skin, muscle, tendon and bone have connective, protective and /or supportive functions.

What amino acid residue is every third residue in collagen? What residue follows it?

Glycine, proline.

Describe the properties of collagen.

• Tropocollagen is the precursor form and is composed of three polypeptide chains about 1000 residues each, in a triple helix rope like coil

• Gly-Pro-X motif

• Hydroxylation is common, lysines and prolines (requires vitamin C)

• cross linked fibrils form after processing

• amount of crosslinking determines rigidity

• 44 genes, 28 types

What disease occurs with a vitamin C deficiency in diet?

Scurvy. Ascorbic Acid (Vitamin C) is a cofactor for prolyl and lysyl hydroxylases that hydroxylate residues in collagen. Melting temperature remains low and skin lesions form, blood vessels weaken.

Describe the mechanism of diffusion of small molecules across lipid bilayers.

Molecules must shed their solvation shell, dissolve in the hydrocarbon core of the membrane and get to the other side of the membrane and be resolvated. This is very difficult for charged molecules. The water shell is not happy.

The longer the acyl chain the ___ fluid the membrane.

Less

The greater the number of double bonds the ____ fluid the membrane is.

More

Provide a brief overview of cholesterol.

Known as the disruptor. Except for on hydroxyl group, the whole thing is hydrophobic. Plasma membranes have more than intracellular membranes. It stiffens membranes.

Briefly describe integral membrane proteins.

Interact extensively with hydrocarbon chains of membrane lipids. Span the bilayer (most).

Briefly describe peripheral membrane proteins.

Proteins bound to membranes primarily by electrostatic and H bond interactions with the head groups of lipids. Cytoplasmic or extracellular, anchored via hydrophobic side chain.

Describe how the primary structure of a protein can contribute to the final localization of a protein in the membrane.

• hydrophobic amino acids can form a helix in the membrane and allowing a channel

• acidic and basic residues are hydrophilic and likely in the cytosolic end

• glycosylation sites are extracellular

What are the types of integral proteins. How many subtypes do they each contain?

• single pass (type I, II, II, TA, small)

• multipass (type I, II, III)

What are some functions of single pass integral proteins?

Integrins, most Golgi apparatus enzymes, P450 enzymes, SNAREs

What some functions of multipass integral proteins?

Most ionotropic receptors, ABC transporters, most GPCRs

What is a lipid raft?

Microdomains of liquid ordered phases within the more dispersed liquid bilayer. Usually enriched in cholesterol, gangliosides, sphingomyelin and membrane proteins. Usually grouped with specific functions (ETC).

WHat are some roles of lipid rafts?

• intracellular signaling (PKC)

• extracellular signal reception (LDL receptor)

• Catalysis (endothelial nitric oxide synthase)

• Structure

What are the six major types of membrane proteins

• Intercellular joinings

• enzymatic activity

• transport

• cell-cell recognition

• anchorage attachment

• signal transduction

Describe a membrane channel

Enable ions to flow rapidly through membranes in a thermodynamically downhill direction (passive transport or facilitated diffusion)

Describe membrane pumps.

Use a source of energy to drive the thermodynamically uphill transport of ions or molecules (active transport).

What is simple diffusion? What two factors determine if it can do this?

Lipophilic molecules can pass through cell membranes because they dissolve in the lipid bilayer.

1. the permeability of the molecule in the lipid bilayer

2. availability of an energy source

What is an antiporter?

A transporter that couples downhill flow of one species to the uphill flow of another (concentration gradient), in the opposite direction of the membrane.

What is a symporter?

Use the flow of one species down its concentration gradient to drive the flow of another species in the same direction across the membrane.

What is a uniporter?

Transport a specific species in either direction, governed only by concentration of that species on either side of the membrane

What is a porin?

A channel built form beta strands and contain essentially no helices

Provide an overview of the passive K+ channel.

• 70 kDA protein, 4 copies come together to form the channel

• On the inside of a cell, the opening is large and allows the K+ ion and its solvent shell in

• the solvent shell goes away and only the K+ ion is allowed to go through the much smaller opening at the other side, interacting with those residues

• Specificity is determined by the selectivity filter and ion size.

What amino acids make up the selectivity filter in the K+ channel?

TVGYG

Why does the K+ channel not interact with sodium?

Even though Na+ is smaller than 1.5 A it is still rejected because it does not lose its water shell. The oxygen in the selectivity filter are positioned so that they do not interact with sodium because the ion is too small, the cost of dehydrating them would be too high.

Why is the K+ channel so fast?

Selectivity filter is highly specific and has 4 binding sites, as ions enter they generate electrostatic repulsion and push each other out.

What are the three types of ATP driven pumps?

P type, ABC transporter, V type proton pump, and F type ATP synthase

Provide a breif overview of the Na+/K+ ATPase.

Generates a concentration gradient such that the cell has a high concentration of K+ and low concentration of Na+ relative to the extracellular space. Hydrolysis of ATP provides the energy needed to actively transport Na+ out of the cell and K+ in. It requires Mg2+, generates membrane potential.

What is secondary transport?

No direct coupling of transport to ATP, instead the electrochemical potential difference is created by pumping ions into and out of the cell is used as energy. Using the favorable gradient to pump against the gradient for another molecule.

Describe cystic fibrosis and nae the gene/protein involved.

• lethal genetic disorder affecting 1:3000 newborns

• accumulation of mucus obstructs airways and digestive tubes

• recurrent infections, inflammation, respiratory failure

• CFTR, an anion channel regulating chloride homeostasis (can’t pump Cl- out of cell)

Where do many CTFR mutations occur?

The nucleotide binding domain (where ATP)

Describe the structure of an ABC transporter.

They are dimers, amino-terminal half of each monomer is the membrane-spanning domain, carboxyl terminal is the ATP binding cassette.

What are the steps to active transport by an ABC transporter?

1. catalytic cycle begins with the transporter free of both ATP and substrate

2. Substrate enters the central cavity of the open form inside the cell (substrate binding increases affinity for ATP binding)

3. ATP binds, conformations change and the two ATP binding domains interact strongly

4. Strong interaction between the ATP binding cassettes changes the overall protein structure, releasing substrate to the cell

5. hydrolysis of ATP releases ADP and a phosphate to reset

What is the most common deletion in cystic fibrosis? What does it do?

F508, makes it so the protein misfolds and does not translocate out of the endoplasmic reticulum to the Golgi. Protein is degraded.

What percentage of CFTR mutations are Class I? What are Class I mutations?

• 22%

• no functional CFTR is made in the first place.

What percentage of CFTR mutations are Class II? What are Class II mutations?

• 88%

• CFTR protein is created but misfolds keeping it from moving to the cell surface

What percentage of CFTR mutations are Class III? What are Class III mutations?

• 6%

• CFTR is created and moves to the cell surface but does not open the channel gate properly.

What percentage of CFTR mutations are Class IV? What are Class IV mutations?

• 6%

• CFTR protein is created and moves to the cell surface but the function of the channel is faulty

What percentage of CFTR mutations are Class V? What are Class V mutations?

• 5%

• Normal CFTR protein is created and moves to the cell surface but is insufficient in quantity

What is a molecular motor?

A device that consumes energy in one form and converts it into motion or mechanical work.

What are some examples of rotary motors in the cell?

ATP synthase, bacterial flagellar motor

What is an example of an ion pump in the cell?

Na/K pump

What are some example of polymerization motors in the cell?

Actin, microtubules, RecA

What are some example of cytoskeleton motors in the cell?

Dynein, kinesin, myosin

What are some example of DNA motors in the cell?

helicases, condensins, viral DNA ecapsidation proteins, bacterial DNA segregation proteins

What are some example of protein remodeling motors in the cell?

unfoldases, refoldases, proteolysis

Provide an overview of ATP synthase.

• a highly conserved enzyme that utilizes the energy from an electrochemical gradient of protons or sodium ions to synthesize ATP

• Produces 40-50 kg of ATP a day

• a mini rotating motor with a bipartite structure consisting of a membrane-bound component (membrane channel) and a water-soluble component (catalytic site for ATP synthesis)

What are the three major types of cytoskeletal proteins?

Microfilaments, intermediate filaments, and microtubules

Briefly summarize tubulin and its role in microtubules.

• microtubules are made of tubulin polymers (alpha and beta)

• Binds GTP (alpha) or GDP (beta)

• protofilaments of tubulin form sheets that roll into tubes as monomers keep binding either side

What does taxol do?

Binds to beta tubulin subunits in a microtubule and prevents depolymerization (freezes cell)

What does Colchicine do?

Causes microtubules to depolymerize and block cell division, binds between alpha and beta tubulin in a dimer.

Describe kinesin.

• little stepper who walks on microtubules away form the - end to the positive end

• has two heads that work in a coordinated way so that they alternatively bind to beta tubulin subunits

• kinesin transport is away from cell center