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Transmembrane proteins span the bilayer
α-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
ECB Fig. 11-36
Bleach with laser beam
If protein is mobile
mobile
then fluorescent
signal moves back into
into
bleached area
Recovery rate measures
mobility
2
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)
ligand)
Non-covalent
interactions
interactions
Binding site
ECB Fig. 4-30
Enzyme (protein)
3
How do enzymes work?
Start by considering free energy
Free energy is amount of useful energy
available to do work
∆G (Delta G) = free energy change
(Reactants - Products)
In a chemical reaction
∆G = ∆H − T∆S
∆H = heat; heat released is negative
∆S = entropy (randomness); increased
randomness is positive
Reactions occur spontaneously if ∆G is negative
Enzymes lower activation energy but have
NO effect on ∆G
Energy of
reactants
Activation
energy
∆G
Energy of
products
ECB Fig. 3-13
3-13
Catalyzed reaction
Uncatalyzed reaction
Enzymes accelerate reaction rates
X
Y
Uncatalyzed reaction
X
Y
Enzyme catalyzed
catalyzed
reaction
ECB Fig. 3-26
4
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
energy
Enzymes can put physical
stress on substrates
Adapted from ECB Fig. 4-35
Thermodynamically Unfavorable Reactions (∆G+)
Y
Many reactions in cells have positive ∆G:
e.g. condensation reactions (forming polymers
reduces randomness so ∆S -, ∆G +)
∆G = ∆H − T∆S
∆G +
Solution: couple to reaction where ∆G (Often hydrolysis of ATP)
X
∆G +
Y
ATP
ADP + P i
X + ATP
∆G -
Y + ADP + Pi +
∆G -
Example of coupled reaction:
synthesis of sucrose
ECB Panel 3-1
∆G values are
additive
5
ATP
(Nucleotide)
ADP + Pi
+ energy
∆G of hydrolysis = -7.3 kcal/mole
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
6
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
O 2, CO 2, N 2, benzene
Small Uncharged
polar molecules
H2O, glycerol, ethanol
Properties of a pure
synthetic lipid bilayer
Large, uncharged
Polar molecules
Amino acids, glucose,
nucleotides
IONS
H+, Na+, HCO 3-,
K+, Ca 2+, Cl -, Mg 2+
ECB 12-2
Transmembrane proteins allow movement of
molecules that cannot move through bilayer
ECB 12-1
But it is not that simple……………
7
Membrane impermeability results in electrical and
chemical gradients across membrane
Charged molecules - transport influenced by concentration gradient
and membrane potential (electrochemical (EC) gradient )
out
Electrochemical
gradient
in
ECB 12-8
Concentration
Concentration
gradient only
only
Conc.
Conc. Gradient
Gradient with
with
membrane
membrane potential
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.
8
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
Channel-mediated
diffusion
(facilitated
(facilitated diffusion)
diffusion)
9
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
(facilitated diffusion
diffusion
down EC
EC gradient)
gradient)
Carrier Proteins:
Active transport
(energy-driven)
(energy-driven)
Transport against EC gradient
Transfer across membrane driven by conformational change in transporter
Slower than channels
Binds transported ligand - highly specific
10
Active transport - three types
-uses energy to drive transport against EC gradient
through carrier protein
ECB 12-9
Coupled transport
Cotransported
Cotransported
Molecule
(against EC
EC gradient)
gradient)
Down EC
EC gradient
gradient
ECB 12-13
SymportSymport- move same
direction
AntiportAntiport- move opposite
directions
Na-Glucose symporter
Move glucose against its EC gradient, using
the energy stored in the Na + gradient.
ECB 12-14
11
ATP-driven pumps
Move
against EC gradient
ATP
Typically move ions generating
EC gradient
EC gradient can then be used
in coupled transport
ADP + Pi
Na+/K+ pump in animal cells
ECB 12-10
Cyclic transport by Na+/K+ pump
Conf.
change 1
Phosphoryation regulates
the enzyme conformation
Low affinity
Na binding sites
High affinity
K binding sites
3
3
3
2
High affinity
Na binding sites
Low affinity
K+ binding sites
2
NaKATPase.avi
Conf.
change 2
2
12
Chemiosmotic coupling of pumps and cotransport
H+ transporters
transporters in
in
vacuole and
and
lysosome are similar
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
13