chapter 4 Flashcards
The shape of a protein is specified by its
amino acid
sequence
Amino acids are connected together by
covalent peptide
bonds
Proteins fold into
3D shapes
Proteins fold into a conformation of
lowest energy
An amino acid contains an
amino group (NH2), a carboxyl group (COOH),
an a-carbon atom and a side chain
covalent link between carboxyl group of one amino acid and amino group of next amino acid
Peptide bond
Linking amino acids together through peptide bonds forms the
polypeptide backbone
polypeptide backbone
a repeating sequence of the core atoms (N-C-C) in
every amino acid
Denature proteins with solvents that
disrupt non-covalent bonds (urea, detergent)
When you remove the solvents from proteins, the proteins will
refold
Protein folding depends on
non-covalent bonds and
hydrophobic interactions
Protein folding occurs in the
aqueous environment of the cell interior
Although chaperones make the folding process more efficient and reliable, the final
3-D shape of the protein is specified
only by its amino acid sequence
What proteins can assist with protein folding
chaperone
Two common secondary structures for folding proteins are
the alpha helix and beta sheet
Amino acid side chains are not or are involved in secondary structure formation
are not
The alpha helix is formed by
by hydrogen bonds in the polypeptide backbone as it
twists, amino acid side groups project out
How many amino acids can form an alpha helix
many
20 __ amino
acids can span a lipid
bilayer, interact with fatty
acids in phospholipids
hydrophobic
Hydrophobic side chains in __ position every __
amino acids form a hydrophobic “stripe”
1st and 4th, seven
Two or more alpha-helices with hydrophobic stripes can
interact to form a
coiled coil
Beta sheets are formed by
by hydrogen bonds
between polypeptide
backbones of adjacent strands
In the beta sheet, the amino acid side chains project…
above & below the
b-sheet
regions of 40-350 amino acids that can fold independently into a stable structure
Protein domains
There are more than __ recognized protein domains
2000
what is the first protein level of organization, primary structure?
amino acid sequence
what is the second protein level of organization, secondary structure?
simple folding patterns (a-helix, b-sheet)
what is the third protein level of organization, tertiary structure?
3-dimensional shape of the polypeptide
what is the fourth protein level of organization, quaternary structure?
multiple polypeptide subunits in a protein complex
Where are protein domains located, what structure?
tertiary
Protein subunits can assemble to form
larger macromolecules
Depending on the location of __ on
the surface of a protein, a single protein subunit
can assemble into a wide variety of shapes
binding sites
Fibrous, elongated proteins are common in the
Extracellular matrix
Elastin and collagen are
ECM proteins
Fibrous, elongated proteins are often stabilized by
disulfide bonds
The binding sites of antibodies are
versatile
All proteins bind to
ligands
powerful and highly specific catalysts
enzymes
bind substrates and hold them in conformations that promote a particular reaction
enzymes
Binding sites for proteins are usually a pocket near
the surface of the folded protein
Binding usually involves many __ bonds
non-covalent
Heme group binds
iron
Heme group facilitates
O2 binding to hemoglobin
Aberrant conformation causes
polymerization
Enzymes are often regulated by other molecules
that bind at a
second site, allosteric control
controls the location,
assembly, stability and activity of protein complexes
covalent modification
can control protein activity by
triggering a conformational change
phosphorylation
regulated by cyclic
gain and loss of a phosphate group
GTP binding proteins
allows motor proteins to
produce large movements in cells
Nucleotide hydrolysis
Z binds to a __ on enzyme and exerts __ control
second, allosteric
As concentration of Z increases, it feeds back to the enzyme that
converts B to X to shut down the pathway
Allosteric control can also provide what feedback
positive feedback
When the concentration of ATP is low, the concentration of ADP is
high
can bind the enzyme, changing its conformation so it favors the
active form
ADP
What protein use ATP to phosphorylate amino acid side chains
Kinases
remove the phosphate group (Pi)
Phosphatases
Pi adds what charge
negative
The __ charge on Pi results in a
conformational change
Phosphorylation can either
activate or inactivate a protein
Ubiquitination stimulates
degradation
Palmitoylation on cysteine targets…
the protein to a membrane
G-proteins with bound
GTP are
active
G-proteins with bound GDP
are
inactive
GTP hydrolysis switches
G-protein
off
GDP-GTP exchange
switches G-protein
on
Many cell signaling
pathways use what protein
G-proteins
what drives
directional movement
NTP (nucleotide hydrolysis)
NTP is observed with
motor proteins
s enables the protein to
bias its conformational
changes in one direction
Nucleotide (NTP) hydrolysis
Amino acids only differ by their
Side chains
breaks holes in cell membranes
Homogenization
Spin the homogenate to enrich for your protein or cellular
component of interest
Centrifugation
2 centrifugation techniques:
Velocity Sedimentation and
Equilibrium Sedimentation
Take advantage of your protein’s unique size, shape, charge or binding properties
Column chromatography
3 chromatography methods:
Gel filtration, Ion exchange and Affinity chromatography
Overview of protein purification
- Centrifugation
- Column chromatography
- Analysis
Assess the purity and identity of your protein sample
Analysis
Assesses the number and sizes of protein in your sample
Gel electrophoresis
Bigger organelles sediment more __ at lower speeds
quickly
1st step differential Centrifugation allows one to make a
Crude, enriched fraction
separates components based on the rate at which they
move through a gradient
Velocity sedimentation
Used to isolate large protein complexes like ribosomes
Velocity sedimentation
separates components based on buoyant density
Equilibrium sedimentation
In equilibrium sedimentation, Components migrate in gradient until their
Density matches that of the surrounding medium
2nd step of Centrifugation
Velocity vs equilibrium sedimentation
Often used to purify DNA, RNA, different membranes
Equilibrium sedimentation
Most soluble proteins are too __ to purify by Centrifugation
Small
Column chromatography can separate
Protein mixtures
Gel filtration separates proteins based on
Size
Beads for gel filtration contain
Small proes
In gel filtration,
__ proteins flow around beads and out the column first.
__ proteins enter the beads and flow out more slowly.
Larger, Smaller
Ion exchange separates proteins based on
Charge
What do yo use for ion exchange
Positively or negatively charged beads and add proteins
In ion exchange,
Positively charged beads will bind
Negatively charged proteins
Positively charged proteins in ion exchange will flow
Through without binding
Increase __ of buffer to elute the protein
ionic strength
In ion exchange, negative ions in buffer….
Displace negatively charged proteins
Affinity chromatography separates proteins
based on
Specific binding
In affinity chromatography, buy beads with a
Covalently attached substrate
Add a protein mixture, wash away proteins that can’t bind t substrate occurs in
affinity chromatography
Analyze protein mixtures by
Gel electrophoresis (SDS PAGE)
How to analyze proteins by gel electrophoresis
- Treat the protein sample with two chemicals
- Load treated protein sample onto gel with electric field
- Proteins migrate towards positively charged anode
- Smaller proteins migrate through the gel faster than larger ones
What two chemicals are used to treat the protein sample in gel electrophoresis
Beta-mercaptoethanol and SDS
What chemical breaks disulfide bonds
Beta mercapotethanol
What chemical denatures proteins & coats them with negative charges
SDS
SDS-PAGE separates proteins by
Size
In SDS PAGE, larger proteins are at the
Top, smaller at bottom
Protein complexes composed of four subunits (2 Heavy Chains, 2 Light Chains)
Antibodies
Small variable __ in antigen binding site recognize and bind antigens in antibodies
loops
In an antibody, Each light chain and heavy chain contains
One variable domain and one or more constant domains
Each antibody contains __ antigen binding sites, which are made up of __
that have variable amino acid sequences
2, loops
These loops in antibodies are located in the ___ of the heavy chain and the light chain on each side of the antibody
variable domains,
To make an antibody, Host animal degrades protein and presents fragments to
antibody-producing cells called
B cells
Binding of antigens stimulates
B cells to divide and secrete antibodies
Use for protein purification
Immunoaffinity chromatography
Use antibodies to detect a particular protein
within a mixture of proteins
Biochemical chromatography
Use antibodies to locate a protein inside a cell
Microscopic detection
In biochemical detection, antigen A is separated from other molecules by
Electrophoresis
In biochemical detection, Incubation with labeled antibodies that bind to antigen A allows…
the position of the antigen to be determined
Flourescent tags of DNA, microtubules, and Actin
DNA= blue
Microtubules= green
Actin=red
Smaller and less dense components
Supernatant
Larger and more dense components
Pellet
1st step differential Centrifugation can’t make what into a pellet
Most proteins
After running the protein through the gel in SDS PAGE..
Stain the gel with dye specific for proteins
common assay for analyzing column fractions
SDS PAGE
Separate denatured proteins according to their size (molecular weight)
SDS PAGE
Separate native (folded) proteins according to their size and shape
Gel filtration chromatography
Separate native proteins according to their ability to bind a specific substrate
Affinity chromatography
Separate native proteins according to their charge
Ion exchange chromatography
Separate proteins according to the rate at which they move through a gradient
Velocity sedimentation
Separate proteins according to their buoyant density
Equilibrium sedimentation
Flourescent microscopy allows you to
Detect specific components
What parts of amino acids are involved in peptide bonds
Amino group on one amino acid and carboxyl group on the other
How do most motor proteins make their movements unidirectional
They couple a conformational change to hydrolysis of an ATP molecule
Binding site of protein structure is located
Inside cavity on protein surface
Variable domains are located near the
Antigen binding site
