Proteins in Solution and in
Membrane
- Proteins exist in solution or embedded in membranes
1
Soluble Proteins
Physical and Chemical Properties of
Soluble Proteins
• The folded conformations of native proteins are
different from that of the unfolded polypeptides in
chemical, physical and biological properties
• Native protein can diffuse and rotate due to their
compactness
• Proteases cleave peptide bonds between domains
or in mobile surface loops
• Functional groups are held in proximity by the
folded conformation
• Only one compact folded structure for each protein
domain
3
Aqueous Solubility
• Wide variations in protein solubility in aqueous solution
– Cytoplasmic proteins are high in solubility: up to 35%
– Structural proteins are essentially insoluble under physiological
conditions
– Many water insoluble proteins are present in membrane
– Insoluble proteins are aggregated into complexes
• The solubility of a protein in water is determined by its
free energy when surrounded by aqueous solvent
relative to in amorphous or solid conditions
4
Interactions of protein molecules with
solvents
• Primarily determined by its surface
• Favorable interactions with water
– Provided by charged and polar groups of the
hydrophylic side chains
• Solubility of a globular protein in water increase
at pH values farther from its PI
• Most proteins can be solubilized in aqueous
solutions by adding detergents
– Detergents unfold the proteins
5
Proteins in Aqueous Solution
• Surrounded by a tightly bound hydration layer
• The bound hydration layer is more ordered and
less mobile than bulk water
– 10% greater density and 15% greater heat capacity
• Interactions of water with proteins
– With the charged groups on the surface of protein
– With the polar groups on the surface of protein
– Monolayer around the protein molecule
6
Salt on the Solubility of Proteins
• Increasing the ionic strength at low values
increase the solubility of a protein –
“Salting-in”
• Solubility of protein decrease at higher salt
concentrations – “Salt-out”
– By increasing surface tension of water by added salt
– Inorganic salts reversibly precipitating proteins
7
Other Solvents and Polymers
• Organic solvents decrease the solubilities of
proteins by lowering the dielectric constant of the
solvent
– Polar interactions are less favorable
• Water soluble polymers (e.g., PEG)
– Volume exclusion: 2 molecules cannot occupy the
same space at the same time
– Unfavorable interactions between a polymer and
charged groups on protein surface
8
Hydrodynamic Properties in
Aqueous Solutions
• Diffusion: due to random rotation and translation
(Brownian motion)
– The rate of translational movement depends on the
size and shape of protein molecule and its interaction
with the solvent
– Frictional rotation
– Bound solvent
9
Sedimentation Analysis
• Sedimentation coefficient (s): the rate at which a protein
molecule sediment in a gravitational field
• Sedimentation rate is proportional to its MW, the
centrifugal force, and the density difference between the
molecule and the solvent
• Svedberg Equation determines sedimentation coefficient
– S of a protein depends upon molecule size, shape, and density
– Density is determined by intrinsic volume of the protein by its van
der Waals volume, surrounding solvent, and interactions of a
protein with other molecules of the solution
Ionization
• Ionization of polar group influence the
folded conformation of proteins
– pH changes of solution
– Charges of side chains changes
– Hydrogen bonds and salt bridges changes
11
Membrane and Membrane
Proteins
Functions of Membranes
• Provide a physical and insulating barrier
between the cell interior and its environment
• Proteins typically compose 50% of the mass (2575% ranges)
• Phospholipids bi-layer
13
Structure of Plasma Membrane (PM)
• The behavior of lipids:
micelles vs. vesicles.
• Phospholipid (PL) bilayer - ~ 5 nm thick,
impermeable to water soluble molecules.
14
Membrane Lipid Molecules - all amphipathic
Phospholipids
serine
Sterols
(cholesterol)
Glycolipids
(sugar lipid)
ECB 11-7
phosphatidylserine
galactocerebroside
Phospholipids
O
Each glycerophospholipid
includes
Š a polar region:
glycerol, carbonyl O
of fatty acids, Pi, & the
polar head group (X)
Š non-polar
hydrocarbon tails of
fatty acids (R1, R2).
H2C
O
R1
C
O
O
CH
H2C
C
R2
O
O
P
O−
glycerophospholipid
O
X
Sphingolipids
O
CH3
H3C
• Sphingolipids (SPLs) also
have a polar head group
and two non-polar tails but
do not contain glycerol
• Instead, the backbone is
sphingosine, a long-chain
amino alcohol
• Sphingosine is a fatty
amine, a glycerol molecule
is never seen!
• Some derivatives are
Ceramide, Sphingomyelin,
and Glycosphingolipids
• Sphingomyelin has a
phosphocholine or
phosphoethanolamine
head group.
N+
H2
C
H2
C
O
P
−
O
O
CH3
phosphocholine
H2C
sphingosine
Sphingomyelin
OH
H
C
CH
NH
CH
O
C
fatty acid
R
HC
(CH2 )12
CH3
Plasma Membrane - lipid bilayer organization
a.
b.
c.
d.
Hydrophobic fatty acid tails on inside
Hydrophilic fatty acid heads on outside
Viscous fluid: allows PLs and proteins to diffuse laterally within PM
Fluid mosaic model:
Rafts inhibit lateral mobility
Flippase enzymes catalyze flipping to other half of bilayer
19
Membrane Fluidity (viscosity)
Describes the physical state of the membrane
Pure lipid bilayer - two states
Liquid state
Hydrophobic tails free to move
Gel state
Movement is greatly restricted
(crystalline gel)
Transition
Liquid at temperatures
Above the transition temp. temperature
Crystalline gel at
temperatures below the
transition temp
Living cells require a fluid membrane, but not too fluid:
Membrane fluidity is regulated by the cell
Membrane fluidity
1. Governed by FA length and saturation
2. Fatty acid length - shorter the FA, the lower the transition
temperature (melting point), favors liquid state
3. Fatty acid saturation - the more saturated, the higher the transition
temperature, favors gel state
4. Presence of cholesterol - broadens the temperature over which
transition occurs.
Melting points of 18-carbon Fatty Acids
Fatty Acid
Stearic acid
Oleic acid
α-Linoleic acid
Linolenic acid
Double bonds
0
1
2
3
Melting point (˚C)
70
13
-9
-17
Lipid Rafts
• Dynamic regions of the plasma membrane enriched in cholesterol, sphingomyelin,
glycolipids, GPI-anchored proteins and some membrane proteins.
• Important for signaling.
• Important as sites for entry and egress of viruses.
• Markers for clathrin-mediated endocytosis are not present in rafts.
22
Plasma Membrane: Functions
• Defines the boundary of the cell and isolates the cell.
• Acts as a selective barrier - maintains composition of cytoplasm, which
is very different from extracellular space.
• Mediates the interaction of the cell with its environment.
• Generates membrane potential.
Mammalian Cell Intracellular and Extracellular Ion Concentration
Ion
Intracellular
Concentration
(mM)
Extracellular
Concentration
(mM)
Cations
Na+
K+
Mg+2
Ca+2
H+
5 - 15
140
0.5
1 x10-5
7 x 10-5
145
5
1-2
1-2
1-2
Anions
Cl-
5 - 15
110
23
Membrane potential
•Many cells have a charge imbalance across the
membrane, typically excess negative charge inside
the cell, and excess positive charge outside (due to
different Na+ and K+ inside and outside cell).
•This charge imbalance results in a voltage across the
membrane.
•Typical animal cells have a membrane potential of
about 70 mVolts, due to difference in Na+ and K+
inside and outside cells.
Lipid Bilayer Permeability
• Membranes are
permeable to
small non-polar
uncharged
molecules
(includes O2 and
N2 and CO2).
• Impermeable to
ions and most
other watersoluble
molecules; these
need specific
transporters to
get across the
membrane.
Transport across the PM - Small molecules
1. Passive diffusion: no MP involved. Small hydrophobic molecules.
2. Facilitated diffusion: mediated by MP, but not energy-dependent.
e.g.: glucose and amino acids (via carrier proteins) and charged ions such as
H+, Cl-, Na+, Ca+ (via channels).
3. Active transport: transport against concentration gradient, driven by ATP
hydrolysis. e.g.: Na+-K+ pump, Ca+ pump.
Uniport - specific for one type of molecule.
Symport - transports 2 molecules together.
Antiport - transports 2 molecules in opposite directions.
Active Transport
Active transport requires energy (ATP hydrolysis).
Can work against a concentration gradient.
Example of active transport:
Na+/K+ pump (Na+ conc is higher outside cells).
3 Na+ ions bind to transporter protein inside cell.
ATP phosphorylates protein, causes conformational change.
The 3 Na+ ions are released outside cell; 2 K+ ions bound.
Triggers dephosphorylation of protein.
Protein goes back to original state; K+ released inside cell.
This is an “antiport”; two ions moving in opposite directions through
the some transporter.
Na+/K+ pump in animal cells
ECB 12-10
Membrane Proteins
α helixor β barrel
Peripheral membrane proteins
Associated with membrane, but not in bilayer
Lipid anchored proteins
Transmembrane proteins span the bilayer
a-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
Functions of integral membrane
proteins
Porins
• Relatively simple transporters located in bacterial outer membranes,
mitochondria and chloroplasts.
• Porin proteins are trimeric, a group of 3 beta-barrels.
• Has a 16-stranded beta-barrel structure
• Core of barrel has narrow aqueous channel.
• Porins are unusual membrane proteins in not very hydrophobic and
in being composed of beta structure
• Small molecules with MW less than about 600 can pass through.
Ion channels
• Found in neurons and other eukaryotic proteins, as well as bacteria
• A well-known ion channel is the potassium channel (bacterial).
• Allows potassium to pass, but not sodium.
Dynamic Behavior of Proteins in
Membranes
• Membrane proteins generally diffuse rapidly in
the 2D plane of the membrane (10-10 cm2/s)
• No flip between the two surfaces
• Proteins in membrane induce disorder in the
lipid layer and restrict the diffusion of
neighboring lipid molecules
• Proteins in membrane interact with each other
more than do proteins in solution
38
Mobility of transmembrane proteins
ECB Fig. 11-36
Bleach with laser beam
If protein is mobile
then fluorescent
signal moves back into
bleached area
Recovery rate measures
mobility
How does a protein get to the correct
cellular location?
• Membrane and organelle proteins contain targeting
(sorting) signals in their amino acid sequence.
• Targeting signals are recognized during or after the
protein is translated
• Special machinery recognizes the signal and
translocates the protein to its correct location
All proteins encoded by nuclear DNA are first
translated on free cytoplasmic ribosomes
• Soluble proteins and proteins targeted to the
mitochondria, chloroplasts and peroxisomes are
completely synthesized on free ribosomes
• Translation of Integral membrane proteins, secreted
proteins, and proteins in the ER, Golgi, and
lysosomes are synthesized on ribosomes bound to
the ER membrane
Proteins that are targeted to the nucleus,
mitochondria, chloroplasts and
peroxisomes are synthesized on free
ribosomes as soluble polypeptides
Translation of secretory mRNA begins
on free ribosomes
• N-terminal signal
sequence emerges from
ribosome tunnel
• Signal recognition
particle (SRP) binds to
the emerging signal
sequence from the
ribosome
SRP/SRP receptor dissociates
from signal sequence
• Ribosome binds to
translocon
• Signal sequence binds to
translocon. Translocon
gate opens
• Signal sequence inserts
into translocon central
cavity w/ N-terminus
toward cytosol
• Polypeptide chain
elongates; signal
sequence cleaved
and degraded in ER
lumen
• Peptide chain
elongation extrudes
protein into ER lumen
SRP receptor initiates the interaction of signal
sequences with the ER membrane
•
Receptor is an α,β dimer – β subunit is an
intrinsic membrane protein
• α-subunit initiates binding of ribosome –
SRP to ER membrane
Ribosome dissociates and is
released from membrane when
protein is completed
How do intrinsic membrane
proteins get inserted into the ER
membrane?
Most cytosolic transmembrane proteins have an Nterminal signal sequence and an internal topogenic
sequence
Type I protein
A single internal signal-anchor sequence directs
insertion of single-pass Type II transmembrane
proteins