Lecture 2

Question 1

ALL amino acids have a ______-

Answer 1

Carboxyl group

Question 2

Do amino acids have a phosphate group naturally?

Answer 2

NO, they need to be phosphorylated

Question 3

Primary structure

Answer 3

Polypeptide chain

Just a string of amino acids

These chains have POLARITY (direction; ends of chain differ)

Question 4

Secondary Structure

Answer 4

Either alpha helix or beta sheets

H bonds determine secondary structure

ONLY have h-bonds

Question 5

Tertiary structure

Answer 5

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

Question 6

Quaternary structure

Answer 6

Many proteins require >1 chain of amino acids; hemoglobin requires 4 (2 alpha, 2 beta) polypeptide chains; heterotetramer (4 different chains)

Question 7

Allosteric Enzyme

Answer 7

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

Question 8

Phosphorylation

Answer 8

Can control protein activity by causing a conformational change
-Covalent modifications also control the location and interaction of proteins

Question 9

Regulatory GTP-binding proteins are switched on and off by the gain and loss of a

Answer 9

Phosphate group

Question 10

Proteins often for large complexes that function as

Answer 10

Machines

Question 11

Feedback Inhibition

Answer 11

Can trigger a conformational change in an enzyme

Question 12

Active Site

Answer 12

Substrate (whatever will CATALYZE the reaction) will bind here

Reaction OCCURS here

Question 13

REgulatory site

Answer 13

This is where a ligand will bind and change the conformation of the protein

REGULATES the protein; activators or repressors bind here

Question 14

The binding of a regulatory ligand can change the _____ between 2 protein conformations

Answer 14

Equilibrium

Question 15

Protein phosphorylation

Answer 15

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

Question 16

DEphosphorylation

Answer 16

Removal of a phosphate by PHOSPHATASE

Question 17

3 amino acids commonly phosphorylated

Answer 17

Serine
Threonine
Tyrosine

ALL of these have an acetyl group to which the phosphate attaches

Question 18

Protein phosphorylation can modify the activity and _____________ in the same direction

Answer 18

Does NOT always go

Question 19

The modification of a protein at multiple sites can control the protein’s

Answer 19

Behavior

Question 20

Proteins can be modified in many different ways and

Answer 20

Locations

Question 21

Ways to modify a protein:

Answer 21

Methylation

Glycolysis

Phosphorylation

Question 22

Many different GTP-binding proteins function as molecular switches

Answer 22

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

Question 23

how proteins are controlled

Answer 23

Allosteric enzymes

Modification by chemical group

GTP binding

Involved in large complexes

Question 24

How proteins are studied

Answer 24

Proteins can be purified from cells or tissues

Question 25

In order to study proteins, must first

Answer 25

break open cell

Question 26

how to decide which method to use to break open cell:

Answer 26

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

Question 27

Cell breakage and initial fractionation of cell extracts

Answer 27

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

Question 28

Centrifugation

Answer 28

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)

Question 29

Differential Centrifugation

Answer 29

Spin homogenate at increasing speeds

Proteins settle in pellet at bottom

Separates on the basis of SIZE and DENSITY

Question 30

Equilibrium Sedimentation

Answer 30

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

Question 31

Protein separation by Chromatography

Answer 31

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

Question 32

Gel-filtration Chromatography:

Answer 32

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)

Question 33

Ion Exchange Chromatography:

Answer 33

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)

Question 34

Affinity Chromatography:

Answer 34

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

Question 35

Affinity chromatography can be used to

Answer 35

Isolate the binding of a protein of interest

Question 36

Protein separation by electrophoresis

Answer 36

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 -

Question 37

Isoelectric Focusing

Answer 37

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

Question 38

2D PAGE

Answer 38

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

Question 39

X-Ray Chrystallography, NMR, microscopy

Answer 39

Used to determine PROTEIN STRUCTURE

Don’t need to know any more than function