Cell Bio Exam 1 Tools and Proteins

Protein Kinase C

Attaches Phosphate to Proteins- Catalytic and regulatory proteins. Catalytic is Kinase. Regulatory is C1 and C2. Amphitropic peripheral protein.

C1 Domains

Activated by Dicylglyercol- moves psuedosubstrate out of PKC’s (or other proteins) active site. DG is found near where…?

C2 Domains

Activated by rise in Ca2+ ions- causes protein to associate with membrane. (In this case, the protein is PKC)

Domains

Single polypeptide chains which fold independently of each other and often provide a unique function. Equal mix of Hydrophilic vs Hydrophobic parts

IDR

Regions of intrinsic disorder. Not an equal mix of hydrophilic and hydrophobic parts, so do not fold into globule. Link domains but can also serve functions if they gain predictable structure (whats an example of this?)

What needs its IDR for binding?

Regulatory protein p27 binds to cyclin/cdk with the help of its IDR. It NEEDS the IDR to bind- when they bind, the IDR gains predictable shape.

p27

Regulatory protein. Binds cyclin/CDK and prevents cell cycle progression. Needs IDR to bind to cyclin/CDK- example that IDR is not useless.

Primary structure

Amino acids thru peptide bonds.

Secondary structure

Alpha Helixes and Beta sheets from hydrogen bonds of the peptide bonds. can be denatured by heat.

Tertiary Structure

Noncovalent (Ionic, hydrogen, nonpolar) bonds between R groups on polypeptides that result in a 3D shape. Can be denatured by heat- and charges can be changed thru pH of environment

Quatanary structure

If equilibrium heavily favors a protein-protein interaction, such that proteins are hardly alone. “subunits'“. Ex: hemoglobin

Structural complimentarity

Complementarity of shape. Useful bc it enhances chemical complimentarity by optimizing distances btwn proteins and providing more surface area for bonds

Chemical complimentarity

Enhanced by structural complimentarity. What’s really important- without chemical complimentarity, structural complimentarity useless. Matching charge + hydrophobicity

Examples of chemical + structural complimentarity

Lipid transport protein carries phospholipids out of membrane- barrel shape for lipid tail to fit into (structural), but inside of barrel is also hydrophobic, which phospholipids like. Also hydrophilic at phospholipid heads.

CAP (catabolic activator protein) only works to activate Lac operon in presence of cAMP- needs to bind.

Fit well together but specific amino acid residues, ligand binding residues, keep it in place. Also has one ionic bond (ionic bonds can be changed by changing pH)

Tool: SDM

Site-directed mutagenisis. Change using PCR primers to force basepair switch- new amino acid. Can be used to test what happens if ligand binding residues are disrupted (which nature does not do)

Protein example with highly conserved ligand binding residues

Cytochrome C- conserved across bacteria to primates. Usually Cys 14, Cys 17, His 18

A proteins PkA is 6.5 and pH is 9. The protein is…?

Deprotenated (No H+)

Aquaporin 3

Allows glycerol, water, and other uncharged cells across membrane. Example for pH changing a proteins binding sites affinity for ligands. At pH 6.5 or higher, Histidine 180 is deprotinated, which allows for ligand recognition.

Prosthetic groups

Non-protein molecule which is essential for a protein to perform its function. Attached permanently, very tightly.

Heme in Hemoglobin

An example of a prosthetic group. It’s the iron containing group which carries oxygen

Disulfide bonds

R-S-S-R. Between thiol groups of two Cysteine. ONLY R-R interactions that are covalently bound. Cannot be broken thru heat, need reducing agent.

Tool: Detergents

Solubatize plasma membrane, replaces lipids around integral protein to shield hydrophobic parts from water

Tool: Salt

Can be used to dissociate peripheral proteins from plasma membrane. Salt competitively inhibits the electrostatic interactions which peripheral proteins use to cling on to lipid membrane or intrinsic proteins

Tool: EDTA

Ethyaline diamine tetra-acetic acid is a mg2+ Ca2+ ion chelator. Mg and Ca can help proteins bind- when ions are taken away, affinity is lost and peripheral proteins disassociate.

Transmembrane proteins

Use hydrophobic alpha helixes to anchor self to membrane. As a rule of thumb, always a glycoprotein. Oligosaccarides attached usually to exoplasmic side. Can be single pass (1 helix) or multi pass (multiple helixes up and down membrane.) Alpha helices are visible thru a ~20 AA strongly hydrophobic patch

Monotropic proteins

Intrinsic protein which does not cross membrane. “One side only.” Ex: Synaptobrevin, COX-2

GPI anchored proteins

Monotropic proteins anchored using glycocyl phosphtidyl intisol. Always exoplasmic, always on c terminus. Glycocyl = sugars link protein to phosphtidyl insitol, which is a phospholipid. phosphytidl insitol is inserted into plasma membrane

Myrisoylated Proteins

Monotropic intrinsic proteins attached to lipid membrane using simple myristyl (kind of lipid) anchor. Attaches at N terminus. Cytosolic side only.

Farnesylated Proteins

Monotropic proteins attached to the plasma membrane using farnesyl groups. C-terminus. Cytosolic side only. Added by farnesyltransferase.

Tools: SDS

Sodium Dodecyl Sulfate. A STRONG detergent that solubitizes everything.

Tool: SDS-Page

“Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis”. Solubitizes membrane and then sorts proteins by molecular weight. Higher weight = more kB.

Amphitrophic proteins

Can associate with membrane, but doesn’t always have to. Can be integral or peripheral depending on protein.

Recoverin

Amphitrophic protein from retina. In presence of calcium, myristyl tail extends and inserts itself into plasma membrane.

Alpha + Beta Spectrins

Peripheral proteins found after SDS-Page of erythrocyte “ghosts”. Structural proteins essential for the concave shape of RBC, which is important bc higher surface area = better gas exchange.

Tool: Coomassie Blue

Stains proteins in SDS page gel blue. Visible to naked eye.

Tool: Sypro Orange

Stains proteins in SDS page orange under UV light.

Tool: Lectins

Bind to oligosaccarides. When tagged with floresence, they can show you where the glycoproteins are on an SDS-Page

Tool: Trypsin

A protease. Does not cross membrane. Eats proteins on side it is introduced on. If protein largely diappears = peripheral. If protein has lower kB on SDS PAGE after - it has a part on that side. If eats on one side and also eats on its “flipped” side, you have a trans membrane protein. Helps determine topology. Usually eats in the spaces in between domains, does not eat domains (tightly folded- harder to get to)

Beta barrels

Transmembrane proteins composed of lots of repeats whcih twist to create cylinder- hydrophobic residues outside, hydrophilic residues inside. Porins and nutrient transport.

Tool: PIPLC

PI-specific Phospholipase C. Can cleave GPI-anchored proteins at the phosphotidyl insitol bond. Once it leaves the membrane, it cannot come back.

Tool: Radiotracer labeling

For membrane topology. Can either use Galactose oxidase (labels galactose in oligosaccharides) or lactoperoxidase (labels tyrosine polypeptides). Can show you what is exposed on what side.