Biology 200

Protein Folding

Membrane Permeability● cells are semi-permeable● permeability is influenced by lipids, proteins, and carbs
Membrane permeability is dependent on the properties of the molecules trying to enter (see image __)● gases and hydrophobic molecules diffuse free across lipid bilayers● small uncharged polar molecules diffuse fairly well across lipid bilayers● diffusion of large uncharged polar molecules across lipid bilayers is negligible ● charged substances (ions) cannot diffuse across the lipid bilayer
(Please Check Unit 2 Review/Videos for Structures of Proteins)
The Quaternary structure is: (FROM POLL)● Stabilized by a combination of hydrophilic or hydrophobic interactions, or both, between polypeptides● Stabilized by all factors stabilizing the tertiary structure of any polypeptide● The three dimensional configuration of different polypeptides making up a molecular complex consisting of several units
Summary of Protein Structures● Primary: linear amino acid sequence of peptide-bonded amino acids (determines the 3D structure)● Secondary: local 3D structure stabilized by backbone H-bonding of the peptide (alpha-helices and beta sheets)● Tertiary: Overall 3D structure (fold) of entire polypeptide; stabilized by side-chain interactions (non-covalent and disulphide bonds) as well as interactions between side chains and backbone atoms● Quaternary: 3D arrangement of polypeptides in a protein composed of multiple subunits; similar stabilization as tertiary.
Main Points of Protein Structure: ● Proteins are assembled in discrete levels (driven to fold by the thermodynamics of the system)● Each level is stabilized by specific interactions and bonds between specific parts of the protein● Proper function of a protein requires proper folding (STRUCTURE DICTATES FUNCTION RAWR)● The chemical properties of the polypeptide chain (each amino acid) determines how the protein folds● Each folding level is thus dependent on the primary sequence for proper folding and proper function (make sure you also consider the environmental conditions that the protein is folding in)

Types of Membrane Proteins

Integral Proteins:● Proteins directly attached to the membrane; amphipathic. Can be monomeric or multimeric● Asymmetry: the orientation of transmembrane proteins matter; the leaflet of attachment matter
Peripheral Proteins:● bound to membrane surfaces through non-covalent association with other membrane proteins● Asymmetry: Different proteins attach to different sides
Transmembrane proteins pass through a lipid bilayer● Transmembrane domain: part of a membrane protein that passes through the lipid bilayer● Most transmembrane domains are alpha helices, though some are beta barrels
Can FRAP tell us about integral vs peripheral proteins?● FRAP can only tell you about the morbidity of a tagged molecule in the lipid bilayer, but not how they are attached● Really just cannot tell without more testing and data = THEREFORE NO (X)
The Membrane is Asymmetric: this means the two halves (leaflets) on the membrane are different from each other● lipid: composition of outer leaflet is different than that of inner leaflet of the bilayer● Proteins: associated with the membrane (both peripheral and embedded) also have specific orientation within the membrane● Carbohydrates: are only found on the non-cytosolic side
Flippases: enzymes in the membrane that move phospholipids from one leaflet to the other(note: flippases/floppases = uni-directional movement; scramblases: bi-directional movement)Glycocalyx: protects the cell, acts in cell communication/identification
Carbohydrates are only attached to the non-cytosolic side (always): this is due to the fact that carbs are added to lipids and proteins in the Golgi and the orientation of those sugars are maintained during transport. Glycocalyx is also known as the sugar coat on the plasma membrane.

How Do We Study Membrane Proteins (and their asymmetry)

A. Bioinformatics Approach: hydropathy plots- to predict the number of transmembrane segments and orientation B. Protein Purification and Biochemical Experiments-to determine membrane protein type, components, and orientation
● How can we predict if a protein is embedded in a cell membrane? Hydropathy plots yayyyyyy!(e.g.) Human protein, Glycophorin A (GYPA)
Hydropathy Plots:● Bioinformatics tool to predict alpha helical transmembrane domains● Computer generated● Peaks above the dotted line (threshold) indicating potential transmembrane alpha-helices● above the dotted line indicates a region that is hydrophobic enough to pass through a membrane.● The span of amino acids also has to be at least 20 amino acids long to pass through a membrane
Examples will be added to Unit 2 Review :)

Protein Purification and Biochemical Experiments

Step 1: Isolate your protein ● Peripheral vs. Integral are differentHow? ● Isolate all proteins from cells grown in culture (in vitro)● Open the cells to collect the proteins
To study isolated proteins, looking at them as individuals, you must also look at the solubility.Peripheral Proteins: Attached IN-directly to membrane by non-covalent interactionshigh salt tends to weaken protein-protein interactions by disrupting electrostatic bondsIntegral Proteins: are held by Van der Waals associations; to remove integral proteins, use detergent with HARSH treatments to make them soluble (ouch >_<)
Step 2: Unfold/Linearize portions by adding SDS and break disulphide bonds
Step 3: Separate Proteins by size with a polyacrylamide gel matrix (SDS-PAGE)Larger molecules are closer to the top of the gelSmaller molecules are closer to the bottom of the gel The SDS added gives the protein an overall negative charge, drawing out the anode (linearization)