Membrane Proteins (Course Pack 9)

What three general categories of membrane proteins are we talking about in class?

-Integral membrane proteins -lipid anchored proteins -peripheral membrane protein

Provide examples of the types of proteins that fall into the integral membrane protein category:

-cell surface receptors -glycoproteins -transmembrane anchors -channels and pores -transport protein

What does it mean to be "associated" with the membrane?

A membrane protein can interact with the membrane in many ways, not necessarily by being "embedded" in the membrane. Proteins can stick to the outside of the membrane (to the polar/charged head groups of the bilayer) without any contact with the interior of the membrane at all, or they can be "stuck" to the membrane by having a covalently attached fatty acid or lipid which inserts into the bilayer. All proteins that are in contact with the bilayer, no matter how they do it, are "membrane associated".

As a graduate student working on your PhD thesis, you must figure out how to purify a membrane protein from tissue culture cells. As a first step, you must "dissolve" the membrane and associated proteins so they are water soluble. How would you do this?

Add detergent if the protein is an integral membrane protein or a lipid anchored protein because you must solubilize the membrane components to solubilize the proteins. The integral membrane protein has hydrophobic stretches that must be part of detergent micelles and the lipid anchored protein has hydrophobic "lipid parts" that must be included in a detergent micelle.

Integral membrane proteins have secondary structures within the protein that allow them to interact directly with the phospholipid tails of the membrane. Describe those secondary structures and how they interact with the hydrophobic phospholipid tails(use pictures to support your description). Second:

Amphipathic alpha-helix: in this case, the alpha-helical transmembrane has alternating polar/charged and nonpolar amino acids to that the amino acids on the side of the helix in contact with the fatty acid tails are nonpolar and the amino acids on the side of the helix that will face a water filled pore are polar/charged. 5 amphipathic alpha-helixs: -O- -O+ H2O +O- -O O- H2O

What does it mean to be an "amphipathic" protein and what does that have to do with being a membrane protein?

An amphipathic protein has stretches of nonpolar amino acids (long enough to span a lipid bilayer) flanked by polar/charged amino acids. The nonpolar region of the protein spans the membrane with its nonpolar amino acid side chains interacting chemically with the fatty acid tails of the membrane and the polar/charged amino acids on the outside of the membrane interacting with water.

Draw a picture detailing the interaction of a peripheral membrane protein with the membrane. Include in your drawing as many possible ways it could happen. What sort of chemical interactions or bonding is responsible for this interaction?

Electrostatic (hydrogen bonding and ionic bonding).

Integral membrane proteins have secondary structures within the protein that allow them to interact directly with the phospholipid tails of the membrane. Describe those secondary structures and how they interact with the hydrophobic phospholipid tails(use pictures to support your description). First:

Hydrophobic alpha-helix: proteins with hydrophobic alpha-helical transmembrane domains; the 20-30 amino acids that span the membrane form an alpha-helix, with the hydrophobic side chains facing the fatty acid tails

How would you isolate a peripheral membrane protein from the membrane? Would you need a detergent?

No detergent needed.... Just break open cells, add salt (like NaCl), spin down membranes in centrifuge and the peripheral membrane protein will be released from the membrane. Membranes will pellet to bottom of tube, peripheral membrane protein will be in aqueous supernatant.

What distinguishes an integral membrane protein from the other two?

Some portion of the integral membrane proteins DIRECTLY interacts with the fatty acid tails of the membrane. Thus, the integral membrane protein is EMBEDDED in the phospholipid bilayer. Lipid anchored proteins (the protein part) and peripheral membrane proteins do NOT interact with the interior of the membrane

How are lipid anchored proteins(meaning, the "protein" part of a lipid anchored protein) and peripheral membrane proteins similar? How are they different?

They are both water soluble proteins lacking appreciable hydrophobic character. Lipid anchored proteins have a COVALENTLY bonded fatty acid (fatty acid modified proteins) or phospholipid (GPI-anchored proteins) that inserts into the phoshpholipid bilayer and anchors the proteinin the membrane

How do the secondary structures described in #6 dictate the functions of those membrane proteins who have them?

a) hydrophobic alpha-helical transmembrane domains: proteins with this type of secondary structure function as receptors, transmembrane anchors, transport proteins. These are proteins that are embedded in the membrane but do not function as pores/channel b) amphipathic alpha-helical transmembrane domains: proteins with this type of secondary structure can form water filled channels in the membrane so long as there are at least 3 alpha helices coming together to form the channel. The hydrophobic sides of the each helix face the fatty acid tails of the membrane and the polar/charged sides of each helix face the interior of a water filled channel. These channels allow water soluble ions to pass through the membrane c) beta-barrel transmembrane domain: proteins with this type of secondary structure form large water filled pores in the membrane with their hydrophobic amino acids facing the fatty acid tails of the membrane and the polar/charged amino acids facing the interior of a large water filled pore. These pores allow larger polar/charged molecules to pass through the membrane.

Integral membrane proteins have secondary structures within the protein that allow them to interact directly with the phospholipid tails of the membrane. Describe those secondary structures and how they interact with the hydrophobic phospholipid tails(use pictures to support your description). Third:

beta-barrel: a beta-barrel is formed from an anti-parallel beta-pleated sheet, with hydrophobic amino acids facing the fatty acid tails of the membrane and hydrophilic amino acid side chains facing the interior of a water filled pore

Describe how lipid anchored proteins (our 2nd protein in the "membrane protein box") associate with membrane

lipid anchored proteins are "water soluble" proteins which have a covalently linked lipid or fatty acid. The lipid or fatty acid part of the lipid anchored protein inserts into the membrane and interacts with the fatty acid tails of the membrane.