Lecture 2 Flashcards
ALL amino acids have a ______-
Carboxyl group
Do amino acids have a phosphate group naturally?
NO, they need to be phosphorylated
Primary structure
Polypeptide chain
Just a string of amino acids
These chains have POLARITY (direction; ends of chain differ)
Secondary Structure
Either alpha helix or beta sheets
H bonds determine secondary structure
ONLY have h-bonds
Tertiary structure
Fold proteins
Domains (subsection of protein) that have certain functions that differ from the rest of the protein; most proteins stop here (only 1 peptide chain); monomer
Quaternary structure
Many proteins require >1 chain of amino acids; hemoglobin requires 4 (2 alpha, 2 beta) polypeptide chains; heterotetramer (4 different chains)
Allosteric Enzyme
When it binds to its ligand, the enzyme of the protein will change conformation; it CHANGES the function of a protein
Have 2 or more binding sites that influence one another
Phosphorylation
Can control protein activity by causing a conformational change
-Covalent modifications also control the location and interaction of proteins
Regulatory GTP-binding proteins are switched on and off by the gain and loss of a
Phosphate group
Proteins often for large complexes that function as
Machines
Feedback Inhibition
Can trigger a conformational change in an enzyme
Active Site
Substrate (whatever will CATALYZE the reaction) will bind here
Reaction OCCURS here
REgulatory site
This is where a ligand will bind and change the conformation of the protein
REGULATES the protein; activators or repressors bind here
The binding of a regulatory ligand can change the _____ between 2 protein conformations
Equilibrium
Protein phosphorylation
Very common mechanism for regulating protein activity
Adding a phosphate group to a protein
Modifies a protein (most common modification method)
Phosphate can be used to turn a protein on or off
Changes overall conformation of protein
PROTEIN KINASE catalyzes
ATP dependent
DEphosphorylation
Removal of a phosphate by PHOSPHATASE
3 amino acids commonly phosphorylated
Serine
Threonine
Tyrosine
ALL of these have an acetyl group to which the phosphate attaches
Protein phosphorylation can modify the activity and _____________ in the same direction
Does NOT always go
The modification of a protein at multiple sites can control the protein’s
Behavior
Proteins can be modified in many different ways and
Locations
Ways to modify a protein:
Methylation
Glycolysis
Phosphorylation
Many different GTP-binding proteins function as molecular switches
When GTP is hydrolyzed, it gives off a phosphate and becomes GDP (off; different structure = different function); GDP dissociates slowly, lets off a GDP, then GTP comes in and binds fast, becoming an active protein again
GTP ALWAYS function in this manner
how proteins are controlled
Allosteric enzymes
Modification by chemical group
GTP binding
Involved in large complexes
How proteins are studied
Proteins can be purified from cells or tissues
In order to study proteins, must first
break open cell
how to decide which method to use to break open cell:
1) What type of cell? (animal, plant, or bacteria)
- Animal cells have NO cell wall to penetrate => can’t be too harsh of a method
- Plant cells have THICK cell wall => harsh method
- Bacteria have cell wall (thinner than plants) => not too harsh, not too gentle method
2) What type of protein are you looking for?
- Protein complex would need GENTLE method so as not to break the complex
Cell breakage and initial fractionation of cell extracts
1) Cell suspension
2a) Ultrasound (high frequency): Medium-harsh method
2b) Mild detergent: Gentle, used for animal cells
2c) Force cells through small hole using high pressure: Harsher method, use for bacteria or plant cells
2d) Shear cells w/ plunger: Harsh method, use for plant cells
3) Homogenate should contain membrane-enclosed organelles largely intact
Centrifugation
Separates things by WEIGHT: left with pellet and supernate
Swing vials in special machine at extremely high speeds
Used to see what’s inside the cell (AFTER is has been broken open)
Differential Centrifugation
Spin homogenate at increasing speeds
Proteins settle in pellet at bottom
Separates on the basis of SIZE and DENSITY
Equilibrium Sedimentation
AKA Density Gradient
Make a gradient of particles (will settle at different levels, not just the bottom)
Separates on the basis of BUOYANT DENSITY, independent of size or shape
Protein separation by Chromatography
Separates proteins based on characteristics
Usually done in a column
Push sample through column
Based on characteristic you separate by, it will push through to different levels
Gel-filtration Chromatography:
Separates by SIZE
Beads have pores of different sizes; proteins > holes in bead will go FASTER; proteins < holes in bead will go SLOWER (more convoluted route)
Ion Exchange Chromatography:
Separates by CHARGE
Add negatively charged beads, negative proteins will slide past, leaving you with POSITIVELY-CHARGED proteins behind (can work in opposite direction, too)
Affinity Chromatography:
Separates by BINDING PARTNER
You KNOW that a protein INTERACTS with another protein
Order certain beads that link to a certain protein
Put in column, proteins that can interact with protein X will bind to it and the beads
All other proteins that don’t interact will flow through and fall out
You can then find out what kind of complex forms
Affinity chromatography can be used to
Isolate the binding of a protein of interest
Protein separation by electrophoresis
Used to separate proteins by adding electrical currents to separate MACROMOLECULES
Proteins can either be positively or negatively charged, so detergent (SDS) is used to change proteins to the SAME charge (it coats the proteins and makes them all negative)
Apply current, they will all move to the POSITIVE protein
Smaller proteins move FASTER, larger proteins move SLOWER (opposite of gel-filtration because this is a straight shot in both cases)
Can use this same process for DNA, but don’t need SDS step since all is already -
Isoelectric Focusing
Protein has no net charge and will not move in an electric field
Uses pH instead
Proteins are electrophoresed in narrow tube of gel in which a pH gradient is established by a mixture of special buffers
Each protein moves to the pH gradient that corresponds to its isoelectric point and stays there
2D PAGE
Two-Dimensional PolyAcrylamide-Gel Electrophoresis
Used for COMPLEX MIXTURES; combines 2 different separation methods
Can resolve >1000 proteins in 2D protein map
1) Native proteins separated in narrow gel based on INTRINSIC CHARGE using isoelectric focusing
2) Gel placed on gel slab, proteins subjected to SDS-PAGE in perpendicular direction to step 1
Each protein migrates to form a discrete spot
X-Ray Chrystallography, NMR, microscopy
Used to determine PROTEIN STRUCTURE
Don’t need to know any more than function
