membrane proteins

45
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

Upload: obanbrahma

Post on 15-Apr-2017

107 views

Category:

Education


1 download

TRANSCRIPT

Page 1: Membrane proteins

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

Page 2: Membrane proteins

• 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

Page 3: Membrane proteins

• Epithelial growth factor (EGF) domains (red), membrane binding domains (yellow), and catalytic domains (blue and gray).

Page 4: Membrane proteins

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.

Page 5: Membrane proteins

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

Page 6: Membrane proteins

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

Page 7: Membrane proteins

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”

Page 8: Membrane proteins

Transport Proteins Classification System

• Channels and Pores

• Electrochemical Potential-driven Transporters

• Primary Active Transporters

• Group Translocators

• Transmembrane Electron Carriers

Page 9: Membrane proteins

Transport of Glucose via Gated Pore

• Glucose binds inducing a conformational change in the transporter allowing the release of glucose inside the cell

Page 10: Membrane proteins

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

Page 11: Membrane proteins

Transport Proteins: P-Type: Na+-K+ ATPase

• Phosphorylation and dephosphorylation trigger conformational changes that determine the direction the channel opens

Page 12: Membrane proteins

Group Translocation• Coupling transport to an exergonic reaction• Sugar translocation in bacteria• Source of energy is PEP

Page 13: Membrane proteins

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.

Page 14: Membrane proteins

Transport Proteins: Symporters• Symport of Na+ and glucose in the intestinal epithelium

Page 15: Membrane proteins

Membrane Receptors• Integral proteins that trigger a response after binding ligands

• Diverse range of function

• Examples: cell surface interactions, endocytosis, signaling

Nicotinic acetylcholine receptor

Page 16: Membrane proteins

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

Page 17: Membrane proteins

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

Page 18: Membrane proteins

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

Page 19: Membrane proteins

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

Page 20: Membrane proteins

• 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.

Page 21: Membrane proteins
Page 22: Membrane proteins

22

• Converted to active form• Regulates response of rod and cone cells in the retina to light

Page 23: Membrane proteins

Phototransduction cycle in the Rod cell

Page 24: Membrane proteins

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

Page 25: Membrane proteins

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)

Page 26: Membrane proteins

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

Page 27: Membrane proteins

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.

Page 28: Membrane proteins

• 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

Page 29: Membrane proteins

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.

Page 30: Membrane proteins

• 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.

Page 31: Membrane proteins

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

Page 32: Membrane proteins

Amino Acid Distribution in TM Helices

• Analysis of TM helices in more than 20 proteins

Page 33: Membrane proteins

Helix-Helix Interactions

• GxxxG motif is a reflection of close packing of TM helices

Page 34: Membrane proteins

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

Page 35: Membrane proteins

Helix-Helix Interactions

• Polar Clamp

Page 36: Membrane proteins

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.

Page 37: Membrane proteins

Protein folding

Page 38: Membrane proteins

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

Page 39: Membrane proteins

Early model: The helical hairpin hypothesis for folding and insertion of a pair of TM helices

• Buries the nonpolar residues inside the helical pair.

Page 40: Membrane proteins

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

Page 41: Membrane proteins

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

Page 42: Membrane proteins

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.

Page 43: Membrane proteins

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.

Page 44: Membrane proteins

Protein insertion can relieve bilayer stress

Page 45: Membrane proteins

X-ray Crystallography