Chapters 6-12 membranes biochemistry and biophysics

1. 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

2. • 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

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

4. 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.

5. 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

6. 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

7. 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”

8. Transport Proteins Classification System
• Channels and Pores
• Electrochemical Potential-driven Transporters
• Primary Active Transporters
• Group Translocators
• Transmembrane Electron Carriers

9. Transport of Glucose via Gated Pore
• Glucose binds inducing a conformational change in the
transporter allowing the release of glucose inside the cell

10. 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

11. Transport Proteins: P-Type: Na+-K+ ATPase
• Phosphorylation and dephosphorylation trigger
conformational changes that determine the direction the
channel opens

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

13. 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.

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

15. Membrane Receptors
• Integral proteins that trigger a response after binding ligands
• Diverse range of function
• Examples: cell surface interactions, endocytosis, signaling
Nicotinic acetylcholine receptor

16. 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

17. 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

18. 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

19. 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

20. • 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.

22. 22
• Converted to active form
• Regulates response of rod and cone cells in the retina to light

23. Phototransduction cycle in the Rod cell

24. 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

25. 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)

26. 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

27. 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.

28. • 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

29. 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.

30. • 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.

31. 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

32. Amino Acid Distribution in TM Helices
• Analysis of TM helices in more than 20 proteins

33. Helix-Helix Interactions
• GxxxG motif is a reflection of close packing of TM helices

34. 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

35. Helix-Helix Interactions
• Polar Clamp

36. 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.

37. Protein folding

38. 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

39. Early model: The helical hairpin hypothesis for
folding and insertion of a pair of TM helices
• Buries the nonpolar residues inside the helical pair.

40. 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

41. 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

42. 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.

43. 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.

44. Protein insertion can relieve bilayer stress

45. X-ray Crystallography