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Membrane proteins
ECB Fig. 11-4
Membrane proteins have a variety of functions
Association of proteins with membranes
Fig. 11-21
 helix or b barrel
Transmembrane proteins span the bilayer
-helix
transmembrane
domain
Hydrophobic R groups of
a.a. interact with fatty
acid chains
Multiple transmembrane helices in one polypeptide
Nonpolar a.a.
Polar a.a.
Hydrophilic
pore
Membrane transporter for polar or charged molecules
Mobility of transmembrane proteins
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ECB Fig. 11-36
Bleach with laser beam
If protein is mobile
then fluorescent
signal moves back into
bleached area
Recovery rate measures
mobility
Peripheral membrane proteins
(associated with membrane, but not in bilayer)
Lecture 5 (cont’d)
Membrane Proteins
Proteins as enzymes
Binding sites
Free energy
Activation energy, enzyme function
Enzyme mechanisms
Kinetic parameters of enzymes
Proteins as membrane transporters
Enzymes bind substrates
Substrate
(ligand)
Binding site
ECB Fig. 4-30
Enzyme (protein)
Non-covalent
interactions
How do enzymes work?
Start by considering free energy
Free energy is amount of useful energy
available to do work
DG (Delta G) = free energy change
(Reactants - Products)
In a chemical reaction
DG = D  DS
D heat; heat released is negative
DS = entropy (randomness); increased
randomness is positive
Reactions occur spontaneously if DG is negative
Enzymes lower activation energy but have
NO effect on DG
Energy of
reactants
Activation
energy
DG
Energy of
products
ECB Fig. 3-13
Uncatalyzed reaction
Catalyzed reaction
Enzymes accelerate reaction rates
X
Y
Uncatalyzed reaction
ECB Fig. 3-26
X
Y
Enzyme catalyzed
reaction
How do enzymes accelerate reactions?
Enzymes can hold
substrates in positions
that encourage reactions
to occur
Enzymes can change the ionic
environment of substrates,
accelerating the reaction
Lower activation energy
Enzymes can put physical
stress on substrates
Adapted from ECB Fig. 4-35
Thermodynamically Unfavorable Reactions (DG+)
Y
Many reactions in cells have positive DG:
e.g. condensation reactions (forming polymers
reduces randomness so DS -, DG +)
DG = D  DS
DG +
Solution: couple to reaction where DG (Often hydrolysis of ATP)
X
ATP
X + ATP
Y
DG +
ADP + Pi
DG -
Y + ADP + Pi +
DG -
Example of coupled reaction:
synthesis of sucrose
ECB Panel 3-1
DG values are
additive
ATP
(Nucleotide)
DG of hydrolysis = -7.3 kcal/mole
ADP + Pi
+ energy
Enzymes can be
regulated
Inhibitors
can bind to active site
Binding in the active
site can prevent substrate
interaction
Enzymes can be regulated at sites other than
the active site
Example: phosphorylation
Fig. 5-36
ECB 4-41
Lecture 5 Outline
Protein Secondary Structure
Membrane Proteins
Proteins as enzymes
Proteins as membrane transporters (Ch 12 ECB)
Channel
Carrier proteins
Facilitated diffusion
Active transport
Lipid Bilayer
Permeability
Small hydrophobic
Molecules
O2, CO2, N2, benzene
Small Uncharged
polar molecules
Properties of a pure
synthetic lipid bilayer
H2O, glycerol, ethanol
Large, uncharged
Polar molecules
Amino acids, glucose,
nucleotides
IONS
H+, Na+, HCO3-,
K+, Ca2+, Cl-, Mg2+
ECB 12-2
Transmembrane proteins allow movement of
molecules that cannot move through bilayer
ECB 12-1
But it is not that simple……………
Membrane impermeability results in electrical and
chemical gradients across membrane
Charged molecules - transport influenced by concentration gradient
and membrane potential (electrochemical (EC) gradient)
out
in
Electrochemical
gradient
ECB 12-8
Concentration
gradient only
Conc. Gradient with
membrane potential (-)
inside
Ion gradients across the plasma membrane
pH 7.2*
pH 7.4*
Different electrochemical gradient for each ion
Electrical and concentration gradient can be opposite (e.g. K+)
Transport problems faced by cells:
- Need to get an impermeable molecule across the
membrane - going WITH its electrochemical gradient
- Need to get a molecule (permeable or impermeable)
across the membrane going AGAINST its
electrochemical gradient
Solution -- specialized membrane proteins for
transport functions.
Two broad classes of transmembrane
proteins
A. channel protein
ECB 12-3
B. carrier proteins
Conformational change
Transport can be passive or active
electrochemical
ECB 12-4
Channels - Passive transport down
elecrochemical gradient
Impermeable
Channel
protein
ECB 12-4
Channel-mediated
diffusion
(facilitated diffusion)
Channel structure
Aqueous pore due to polar
and charged R groups
ECB 11-24
Always passive transport
Mechanism of K + channel selectivity
ECB 12-7
Carrier mediated
Diffusion
(facilitated diffusion
down EC gradient)
Carrier Proteins:
Active transport
(energy-driven)
Transport against EC gradient
Transfer across membrane driven by conformational change in transporter
Slower than channels
Binds transported ligand - highly specific
Active transport - three types
-uses energy to drive transport against EC gradient
through carrier protein
ECB 12-9
Coupled transport
Down EC gradient
ECB 12-13
Cotransported
Molecule
(against EC gradient)
Symport- move same
direction
Antiport- move opposite
directions
Na-Glucose symporter
Move glucose against its EC gradient, using
the energy stored in the Na+ gradient.
ECB 12-14
ATP-driven pumps
Move
ATP
ADP + Pi
against EC gradient
Typically move ions generating
EC gradient
EC gradient can then be used
in coupled transport
Na+/K+ pump in animal cells
ECB 12-10
Cyclic transport by Na+/K+ pump
Conf.
change 1
Phosphoryation regulates
the enzyme conformation
3
3
3
2
High affinity
Na binding sites
Low affinity
K+ binding sites
2
NaKATPase.avi
Low affinity
Na binding sites
High affinity
K binding sites
Conf.
change 2
2
Chemiosmotic coupling of pumps and cotransport
H+ transporters in
vacuole and
lysosome are similar
Osmosis
Osmosis: movement of water from region of low solute
concentration to region of high solute concentration (or high
water potential to low water potential)
How do cells prevent osmotic swelling?
ECB 12-17