membrane proteins
TRANSCRIPT
Membrane Enzymes
Definition: Enzyme in which the heterogeneity and dimensionality of the membrane affects the activity.
• Diverse catalytic functions
• Many require specific lipids for activity
• Restricted to the available substrate near them in the membrane- not the concentration of the bulk solution
• Can be integral or peripheral
•Very few structures are characterized
• Also called cyclooxygenase (COX), main catalytic function is the conversion of arachidonic acid to prostaglandin H2 (PGH2)•Monotopic integral membrane proteins: bind to luminal leaflet of ER membrane and nuclear membrane•Implications in thrombosis, inflammation, neurological disorders,and cancer.
• Great deal of attention as the target of non-steroidal anti-inflammatory drugs (NSAIDs)
Prostaglandin H2 synthase
• Epithelial growth factor (EGF) domains (red), membrane binding domains (yellow), and catalytic domains (blue and gray).
Structure of a PGHS monomer, showing POX and COX active sites
• Each monomer has 3 domains: catalytic domain (blue) has two active sites the POX (top of heme) and the COX (bottom)
•Arachindonic acid (yellow/green space fill) is bound between these
•Membrane binding domain (yellow/orange) below arachindonic acid
•Epidermal growth factor (green) is on the side that becomes the subunit interface in the dimer.
NSAIDS
•Many NSAIDS act as competitive inhibitors• They prevent substrate binding by occupying the upper part of the
COX channel•Interactions between the drugs and the enzyme are hydrophobic
• Exceptions being the interaction of the acidic NSAIDs with Arg120 and potential of a hydrogen bond with Ser530
Transport Proteins
Definitions:
• Uniport:
• Cotransport:
• Symport:
• Antiport:
• Electroneutral:
• Electrogenic:
One molecule is transported at a time
Tightly couple movement of more than one molecule
Same direction
Opposite directions
No net transfer of charge across membrane
Transfer of molecules creates a charge separation across the membrane
Transport Proteins
Definitions:
• Active:
• Primary:
• Secondary:
• Passive:
Requires energy to pump solutes “uphill”
Hydrolysis of ATP
Symport or antiport coupled ion gradients made by primary active transporters
Does not require energy. Solutes flow “downhill”
Transport Proteins Classification System
• Channels and Pores
• Electrochemical Potential-driven Transporters
• Primary Active Transporters
• Group Translocators
• Transmembrane Electron Carriers
Transport of Glucose via Gated Pore
• Glucose binds inducing a conformational change in the transporter allowing the release of glucose inside the cell
Transport Proteins: ATPases• Primary active transporters
• Two super families of ATPases
• Superfamily 1:
• Superfamily 2:
• P-Type: located in the plasma membrane
• F-Type: located in mitochondria and bacteria
• A-Type: Transports anions
• V-Type: Maintains the low pH of vacuoles in plant cells and lysosomes, endosomes, Golgi and secretory vesicles of animal cells
Transport Proteins: P-Type: Na+-K+ ATPase
• Phosphorylation and dephosphorylation trigger conformational changes that determine the direction the channel opens
Group Translocation• Coupling transport to an exergonic reaction• Sugar translocation in bacteria• Source of energy is PEP
Transport Proteins: Symporters• Lactose Permease of E. Coli• Secondary active transport that uses ion gradients
The proton gradient made by the respiratory chain is used to drive the uptake of lactose by lactose permease.
Transport Proteins: Symporters• Symport of Na+ and glucose in the intestinal epithelium
Membrane Receptors• Integral proteins that trigger a response after binding ligands
• Diverse range of function
• Examples: cell surface interactions, endocytosis, signaling
Nicotinic acetylcholine receptor
Membrane Receptors: G-Protein Coupled Receptors
Ligand binding activates heterotrimeric guanine nucleotide binding proteins (G-Proteins) which transmit and amplify signals by changing the concentration of cAMP
G protein-coupled receptors• GPCRs respond to chemicals, light or odor and activate
G proteins to initiate signal cascades– Prototype rhodopsin– Share a common structure of seven TM helices
• Mechanosensitive (MS) channels transduce physical perturbations of the membrane into chemical and electrical signals
Generalize function of GPCR
•Respond to a variety of stimuli including : light, odorants, calcium ions, small molecules and proteins
•Trigger activation of the αβγ complex which stimulates the release of second messengers
Rhodopsin• located in rod cells of the eye
•Rhodopsin consists of an apoprotein called opsin and a chromophore, 11-cis retinal
•Bovine rhodopsin spans the membrane with seven α-helices with its C-terminus in the cytosol and its N-terminus on the extracellular surface
•The seven helices have highly conserved residues at key positions
• The eye has two types of light sensitive neurons, rod cells and cone cells•Rod cells responsible for high resolution and night vision while cone cells are responsible for discerning color
• Hyperpolarization of rod cells in response to light•In the absence of light cGMP-dependant ion channels in the outer segmant of the rod are open.
• Decreases the Na+ gradient being pumped out by the Na+K+-ATPase.
•Light absorption by rhodopsin causes the degradation of cGMP and the channels close and the cell becomes hyperpolarized.
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• Converted to active form• Regulates response of rod and cone cells in the retina to light
Phototransduction cycle in the Rod cell
Mechanosensitive Transducers
• MS channels respond to mechanical stresses applied to the membrane or to membrane attached elements of the cytoskeleton.– Enables organisms to respond to touch,
pressure, sound and gravity.• Fall into two broad classes depending on
whether cytoskeleton elements are involved
Two classes of mechanosensitive channels in vertebrates
I. “Swinging gate” triggered by stress of cytoskeleton
II. Channel opens due to pressure in the bilayer(osmotic channels)
Bioinformatics
• Purification and crystallization of membrane proteins are complicated by the presence of lipids and detergents
• Must rely on methods for determining primary sequence of amino acids and genomic sequences
• The planar dimensionality and hydrophobicity of the bilayer aides in the prediction of membrane protein topology
Bioinformatics: Predicting Transmembrane Segments
Hydrophobicity scales and plots predict which portions of the sequences are likely to appear in the lipid bilayer based on their primary sequence of amino acids.
• Proves difficult for a number of reasons:
1)many reagents used that were thought to not permeate the membrane, were later discovered
to permeate the membrane
2) many membrane proteins are protease resistance when in membrane
3) Epitope recognition by antibodies can ambiguous
Predicting Orientation of Transmembrane Segments
Predicting Orientation of Transmembrane Segments in bacteria
• Fuse gene with reporter enzymes inserted into predicted loop regions of E. Coli that rely on a specific orientation for function.
• Effective reporter enzymes include PhoA, Bla, and LacZ
• Works well for bacterial proteins, few eukaryotic membrane proteins have cloned into E.coli.
• Identification of glycosylation sites is used to identify which domains or loops are exported from the cytoplasm
• Fusions to green fluorescent protein, which can reveal the fusion protein’s compartmentalization
Predicting Orientation of Transmembrane Segments in Eukaryotes
a) Confocal images of HeLa cells stably transfected with subunit of a translocase in the outer membrane of the mitochondria. b) view of mitochondria GFP labeled translocase subunit. c) Detailed view of mitochondria in negative control cells transfected with inner mitochondrial membrane protein CIII-EGFP. The scale bars in panels (b) and (c) correspond to 5 μm.
Positive Inside Rule• Using topological data obtained on E. Coli and protein info from photosynthetic RC, statistical analysis revealed a prevalence of basic residues in the cytoplasmic loops
This rule helps predict orientation of the TM helices from a.a sequence
Amino Acid Distribution in TM Helices
• Analysis of TM helices in more than 20 proteins
Helix-Helix Interactions
• GxxxG motif is a reflection of close packing of TM helices
Helix-Helix Interactions• Helix-Helix interactions are also stabilized by several types of hydrogen bonding
• One particular pattern that is seen is the“Serine Zipper” motif
Helix-Helix Interactions
• Polar Clamp
Bioinformatics: β-Barrels• β-Barrels cannot be predicted by α-helix methods• β-Barrels prediction models use hydropathy analysis and known characteristics of β-Barrels, such as an even number of β-strands, antiparallel strands, periplamic N and C termini etc.
Protein folding
Protein Folding and Biogenesis
• Insertion of nascent proteins into the membrane involves their translocation out of the cytoplasm by the same export machinery used to secrete proteins.
• In vitro analysis of TM proteins require insertion into a lipid bilayer, micelle or other model membrane
Early model: The helical hairpin hypothesis for folding and insertion of a pair of TM helices
• Buries the nonpolar residues inside the helical pair.
two-stage model, and possible third stages
• Stage 1: describes the insertion of each individual helix driven by the hydrophobic effect and stabilized by hydrogen bonds along the backbone
• Stage 2: assemble by packing together
• Stage 3: binding of prosthetic group, folding of loops, assembly of oligomers
Possible third stage
Stage 1 can be divided into a four-step thermodynamic model
Hydrophobic side chains provide enough free energy for partitioning
Folding to α-helix is likely induced by partitioning.
Entropic costs of insertion are also compensated for by the hydrophobic effect
Other intrinsic factors influence packing
Folding of β-barrels• β-barrels have hydrogen bonds between neighboring strands as
opposed to α-helices• Assumed that all the strands of barrel are formed at the same
time.
Folding studies of TM domain of OmpA
Experiments support a model of initial insertion of a compact pore, followed by slower transversing of the bilayer.
Protein insertion can relieve bilayer stress
X-ray Crystallography