Module 3: Biomembranes and cell architecture Flashcards
Four reasons why scientists purify proteins
- For structural analysis
- For functional analysis
- For determination of amino acid sequence
- Development of antibodies to a protein
Seven steps to purify proteins
- Develop a good assay
- Select protein source
- Break open cells - protein extract
- Solubilize protein
- Stabilize protein
- Fractionate
- Determine purity
Protein assay
- way of detecting presence of specific protein and estimating the concentration of the protein
- the assay should be specific to the protein you are studying and based upon unique characterstics
Protein source and extraction
- easily obtainable in large amounts
- low in proteins that may co-purify
- low in proteases that may destroy the protein of interest ( or can have protease inhibitors instead)
Protein solubilzation
- by changing conditions in your extract, you can change some of the conditions of your protein
- soluble proteins can be affected by the pH of solution, and salt concentration
- insoluble proteins can be affected by the presence of detergents
Soluble proteins
cytosolic protein and secreted protein
Insoluble proteins
transmembrane proteins and membrane-associated proteins
Protein stabilization
proteins of interest must be stable throughout the extraction process
seven parameters of protein stabilization
- temperature
- protease inhibitors
- ligands
- salts
- metal ions
- concentration
- pH
Fractionation
- term used to describe the process of separating proteins into different groups or fractions
- depends on proteins’ size, shape, polarity, charge and solubility
three fractionation techniques based on size
gel electrophoresis, gel filtration chromatography and ultracentrifugation
two fractionation techniques based on polarity
adsorption chromatography and hydrophobic interaction chromatography
two fractionation techniques based on charge
ion exchange chromatography and gel electrophoresis
fractionation technique based on specificity of binding
affinity chromatography
Define differential centrifugation
- common way to begin protein isolation
- this method is not used to isolate a single protein but a subset of proteins based upon their mass and density
Describe how differential centrifugation works?
First tube has tissue cell/extract. Spin it in an ultracentrifuge at 1000G and will get a pellet at the bottom that contains nuclei and chloroplast, while the aqueous supernatant will have everything else. Spin it again (with the aqueous supernatant) and pellet will have mitochondria. Third spinning will have a pellet containing membranous networks within the cell (ER, golgi, lysosomes, perioxisomes). The supernatant remaining will have cytosolic protein.
Define chromatography
- second common step in fractionation
- technique in which an aqueous extract is poured into a column containing a matrix or a slurry of beads
How does chromatography work?
Column contains matrix and valve at the bottom of the column which allows for aqueous protein to move through it. If there are many interactions between protein and the beads then it will move slowly and vv. In this way proteins have been separated based on degree of interactions with the beads in the column
Ion exchange chromatography
beads are charged. Proteins that are the same charge as the beads will flow more quickly because they are being repelled, while oppositely charged proteins will move slowly because they are attracted. To get rid of the oppositely charged proteins , we can wash it using salt solution, warm wash solution or a change in the pH of the wash solution
Gel filtration chromatography
used to separate based on size. Beads have small holes or cavities in them. Small proteins get trapped while large proteins can move through.
Affinity chromatography
used to separate based on specificity of binding to another molecule. Beads are attached to an antibody which will specifically associate with an antigen. Protein molecules that are not recognized by antibody will flow through, and some will form covalent bonds with the antibody making it move slower. Proteins can be removed by changing pH, increasing temperature, and adding salt concentration.
SDS-PAGE electrophoresis
used to separate proteins by intentionally denaturing the protein. SDS denatures proteins and then coats them and all proteins become negatively charged, eliminating shape and charged density. After protein mixtures are loaded onto polyacrylamide gel. By applying a charge across the gel, all the proteins will move towards the positive end of the gel and it will move at different rates based solely on molecular weight.
Western Blot
combination of SDS-PAGE electrophoresis and protein specific antibodies
Mutli-step purification
- Crude cellular extract
- Precipitation with ammonium sulfate
- Ion-exchange chromatography
- Size-exclusion chromatography
- Affinity chromatography
how to measure specific activity
specific activity = enzyme activity/amount of protein (mg)
Light microscopy
uses light to illuminate the object we are looking at allowing us to visualize most cells and organelles inside eukaryotic cells
Electron microscopy
uses beam of electrons to illuminate the object allowing us to visualized small bacteria, viruses, protein complexes, etc.
Resolution
measure of the minimum distance between two objects that can be distinguished from one another. smaller D = resolution is better
Phase contrast microscopy and Nomarski microscopy
- complementary techniques of producing high contrast images of unstained and unfixed transparent biological specimens
- both methods rely on enhancing the inherent difference in density of different regions of specimen but they accomplish it in different ways
Another name for Nomarski microscopy
Differential interference contrast (DIC)
Phase contrast
- tends to favour clear visualization of internal cellular structures
DIC
- provides clearer, sharper images of the edges and surfaces
Advantage of phase contrast and DIC
live specimens can be visualized
Fluorescence and Immunofluorescence microscopy
can be used to reveal the specific location of specific molecules in the cell
Fluorophore
fluorescent molecule attached to secondary antibody so it can help conform the protein and visibly isolate itself from anything else
Gene fluorescent protein (GFP) and how is it used?
- naturally fluorescent protein of jellyfish
- can use recombinant technology to create a gene fusion between the gene coding for your protein of interest and the gene coding for GFP. This gene fusion can then be reintroduced into your organism where gene can be expressed to make fusion protein
Confocal scanning microscopy
technique that allows a researcher to obtain high resolution images from fluorescently labeled samples
Deconvolution microscopy
creates similar image as one created by confocal scanning using traditional fluorescence microscopy
Transmission electron microscopy
beam is directed through a thinly sliced specimen to form an image
stained, dense areas = dark
unstained, sparser areas = light
Scanning electron microscopy
beam of electrons directed at surface of solid protein revealing information about surface morphology and texture
Biomembranes
- define boundaries in a cell by separating the interior from the exterior
- selectively permeable
Plasma membrane
biomembrane enclosing cell
Internal membrane
biomembrane enclosing an intracellular compartment
direct surfaces on biomembranes
cytosolic (faces, inside), exoplasmic (faces, outside), and lumenal (found only in interior of organelles)
What does a basic bilayer structure consist of?
consists of the hydrophobic tails (squiggly lines) and polar head groups (circle)
Basic structural unit of biomembranes
phospholipids
Phospholipids
an amphipathic molecule that will spontaneously arrange themselves, in an aqueous solution, to form a bubble like structure (micelle)
Micelle
- produced when a single sheet of phospholipids assemble with an hydrophobic core and hydrophilic wall
- lowest energy stable state for molecules in this environment
Chemical makeup of the phospholipid bilayer
- diglycerides contain two fatty acids linked to glycerol and have a phosphate group attached to the third hydroxyl group of glycerol, producing a phospholipid.
- fatty acids are hydrophobic while phosphate group is hydrophilic
Three characteristics of biomembranes
- Extremely dynamic and fluid allowing for fusion of biomembranes
- can be deformed without tearing
- allows lateral movement within biomembrane
Leaflet
each layer of phospholipid membrane
FRAP
- stands for fluorescence recovery after photobleaching
- microscopic labelling technique that allows a researcher to track and measure the fluidity of proteins within a membrane
- by bleaching a portion of the molecules, you can notice that those bleached patches are recovered because fluorscently tagged proteins have moved into the bleached area (i.e. fluidity)
characteristics that affect membrane fluidity
lipid composition and temperature
How does lipid composition affect membrane fluidity?
increased order in bilayer results in decreased fluidity. Increased order can result from saturated acyl chains packed tightly or long acyl chains (vv)
How does lipid composition affect temperature
lower temperatures results in decreased fluidity (vv)
Lipid rafts
microdomains in membranes that are composed of the less fluid components
Fluid mosaic model
describes the lateral movement of phospholipids and proteins in membrane leaflets
three classes of membrane proteins
integral membrane proteins (span across the phospholipid bilayer), lipid anchor proteins (anchored in one leaflet) and peripheral proteins (attached to polar surface of membrane or indirectly via other proteins)
Ways in which integral proteins may pass through a membrane
single-pass TM protein, multi-pass TM protein, beta-barrel, and channels
single-pass TM protein
single hydrophobic alpha helix spans phospholipid bilayer, leaving domains on interior and exterior surfaces of membrane
multi-pass TM protein
uses 7 membrane-spanning domain or serpentine domain with 7 alpha-helices that interact with one another to form a transmembrane domain
beta-barrel
formed from 16 beta strands
channels
collection of alpha-helices