Proteins in Solution and in Membrane - Iowa State Universityduahn/teaching/Biomodulation and...
Transcript of Proteins in Solution and in Membrane - Iowa State Universityduahn/teaching/Biomodulation and...
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Proteins in Solution and in Membrane
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Proteins exist in solution or embedded in membranes
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Soluble Proteins
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Physical and Chemical Properties of Soluble Proteins
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• 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
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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
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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
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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
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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
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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
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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
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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
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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
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Membrane and Membrane Proteins
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Functions of Membranes
• Provide a physical and insulating barrier between the cell interior and its environment
• Proteins typically compose 50% of the mass (25- 75% ranges)
• Phospholipids bi-layer
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Structure of Plasma Membrane (PM)
• Phospholipid (PL) bilayer - ~ 5 nm thick, impermeable to water soluble molecules.
• The behavior of lipids: micelles vs. vesicles.
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Membrane Lipid Molecules - all amphipathic
phosphatidylserinegalactocerebroside
PhospholipidsSterols
(cholesterol)
Glycolipids (sugar lipid)
serine
ECB 11-7
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Each glycerophospholipidincludes
a polar region: glycerol, carbonyl Oof fatty acids, Pi, & the polar head group (X)
non-polarhydrocarbon tails of fatty acids (R1, R2).
O P O
O−
O
H2C
CH
H2C
OCR1
O O C
O
R2
X
glycerophospholipid
Phospholipids
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Sphingolipids
• 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.
H2CHC
O
CH
NH CH
C
CH2
CH3
H
OH
( )12
C
R
O
PO O
OH2C
H2CN+
CH3
H3C
CH3
Sphingomyelin
phosphocholine
sphingosine
fatty acid
−
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a. Hydrophobic fatty acid tails on insideb. Hydrophilic fatty acid heads on outsidec. Viscous fluid: allows PLs and proteins to diffuse laterally within PMd. Fluid mosaic model:
Rafts inhibit lateral mobility Flippase enzymes catalyze flipping to other half of bilayer
Plasma Membrane - lipid bilayer organization
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Describes the physical state of the membrane
Pure lipid bilayer - two states
Liquid stateHydrophobic tails free to move
Gel stateMovement is greatly restricted
(crystalline gel)
Transition temperature
Liquid at temperaturesAbove the transition temp.
Crystalline gel at temperatures below the
transition temp
Membrane Fluidity (viscosity)
Living cells require a fluid membrane, but not too fluid:Membrane fluidity is regulated by the cell
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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 AcidsFatty Acid Double bonds Melting point (˚C)
Stearic acid 0 70Oleic acid 1 13α-Linoleic acid 2 -9Linolenic acid 3 -17
Membrane fluidity
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• 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.
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Lipid Rafts
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• 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.
Ion IntracellularConcentration(mM)
ExtracellularConcentration(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
Mammalian Cell Intracellular and Extracellular Ion Concentration
Plasma Membrane: Functions
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•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.
Membrane potential
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• Membranes are permeable to small non-polar uncharged molecules (includes O2 and N2 and CO2 ).
• Impermeable to ions and most other water- soluble molecules; these need specific transporters to get across the membrane.
Lipid Bilayer Permeability
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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.
Transport across the PM - Small molecules
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Uniport - specific for one type of molecule.Symport - transports 2 molecules together.Antiport - transports 2 molecules in opposite directions.
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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.
Active Transport
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Na+/K+ pump in animal cells
ECB 12-10
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Membrane Proteins
α
helixor β
barrel
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Peripheral membrane proteins
Associated with membrane, but not in bilayer
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Lipid anchored proteins
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Transmembrane proteins span the bilayer
Hydrophobic R groups of a.a. interact with fatty acid chains
a-helix transmembrane
domain
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Nonpolar a.a.
Polar a.a.
Multiple transmembrane helices in one polypeptide
Hydrophilicpore
Membrane transporter for polar or charged molecules
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Functions of integral membrane proteins
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• 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.
Porins
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• 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.
Ion channels
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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
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Bleach with laser beamBleach with laser beam
If protein is mobile If protein is mobile then fluorescent then fluorescent
signal moves back into signal moves back into bleached areableached area
Mobility of transmembrane proteins
Recovery rate measuresmobility
ECB Fig. 11-36
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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
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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
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Proteins that are targeted to the nucleus, mitochondria, chloroplasts and peroxisomes are synthesized on free ribosomes as soluble polypeptides
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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
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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
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• Polypeptide chain elongates; signal sequence cleaved and degraded in ER lumen
• Peptide chain elongation extrudes protein into ER lumen
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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
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Ribosome dissociates and is released from membrane when
protein is completed
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How do intrinsic membrane proteins get inserted into the ER
membrane?
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Most cytosolic transmembrane proteins have an N- terminal signal sequence and an internal topogenic
sequence
Type I protein
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A single internal signal-anchor sequence directs insertion of single-pass Type II transmembrane
proteins