1. Types of membrane proteins
Transmembrane proteins
Cytosolic membrane proteins
External membrane proteins
Peripheral membrane proteins

2. Types of membrane proteins
4. Peripheral membrane proteins
Proteins on either side of the membrane that associate with the polar ends of transmembrane proteins by noncovalent (ionic) interaction (Fig )

3. Peripheral Membrane Proteins Noncovalent association with the polar
ends of transmembrane proteins

4. How do transmembrane proteins cross the bilayer?
Most do so in an α-helix conformation
α -helix composed largely of hydrophobic amino acids that interact with the hydrophobic fatty acids of phospholipid (Fig )

5. Hydrophobic amino
acids in light green
& yellow

6. Glycophorin Single-pass transmembrane protein with an α helix
transmembrane domain

7. How do transmembrane proteins cross the bilayer?
Most do so in an α-helix conformation
α -helix composed largely of hydrophobic amino acids that interact with the hydrophobic fatty acids of phospholipid
Peptide bonds are polar
Because of absence of water in an α -helix , peptide bonds driven to form hydrogen bonds with each other. (Fig. 3-9)

8. Arrangement
of atoms showing
H bonds
Ribbon +
C and N
backbone
Ribbon illustration
of α-helix
The N-H of every peptide bond is
hydrogen-bonded to the carbonyl
group of the peptide bond 4 amino
acids away

9. How do transmembrane proteins cross the bilayer?
2. Other proteins traverse the bilayer as an antiparallel β sheet, forming a β barrel
This also allows peptide bonds in a lipid bilayer to hydrogen bond (Fig. 3-9)

10. Antiparallel β sheet
(peptide chains run
in opposite directions)
Peptide chains
held together
by hydrogen bonds
between peptide
bonds of adjacent
peptide chains

11. β-barrel proteins
Some β barrels form transmembrane channels for transport
Very abundant in the outer membrane of mitochondria and chloroplasts & bacteria
They form water-filled pores
e.g. the porin barrel
Formed from a 16 stranded antiparallel β sheet
Rolls up into a cylindrical structure (Fig 10-21)

12. Polar amino acid
residues line the
aqueous channel
on the inside
Nonpolar amino
acid residues
project from the
outside and
interact with the
lipid fatty acid
sidechains

13. Hydropathy plots
Membrane proteins are difficult to crystallize because of their hydrophobicity
Accordingly, very few have been analyzed by X-ray crystallography, the standard technique to resolve molecular structure.

14. Hydropathy plots
However, DNA cloning and sequencing have enabled identification of amino acids of membrane proteins
Structural information can be inferred from this sequence information
Specifically, can predict where in the protein transmembrane segments reside from a determination of hydrophilic and hydrophobic amino acids
Transmembrane segment is typically amino acids with high degree of hydrophobicity (Fig 10-20)

15. Hydropathy Plots 1 membrane-spanning α helix Hydrophobic
7 membrane-spanning α helices
Hydrophilic
Hydropathy Plots

16. Solubilization of membrane proteins
Transmembrane proteins are difficult to solubilize because of their hydrophobicity
Requires detergents
Small amphipathic molecules that form micelles in water (Fig )

17. Cross section of a detergent micelle
SOLUBILIZATION
The hydrophobic ends of the detergent bind to the
hydrophobic region of the transmembrane protein, thereby
displacing lipid molecules
Since the other end of the detergent molecule is polar,
the protein is brought into aqueous solution as a
detergent-protein complex

18. Detergent
solubilization
of membrane
proteins

19. Solubilization of membrane proteins
Transmembrane proteins are difficult to solubilize because of their hydrophobicity
Requires detergents
Small amphipathic molecules that form micelles in water (Fig )
By using detergents, transmembrane proteins can be solubilized and reconstituted into liposomes
Powerful means of analyzing their properties (Fig )

20. Na+- K+ pump
solubilized &
reconstituted into
liposome
(ion pump present
on plasma
membrane of most
animal cells – uses
ATP for energy)
Pumps Na+ out of
the cell and K+ into
the cell

28. Sidedness of the Red Cell Membrane
Easiest plasma membrane to isolate – hence well studied
Reason:
No internal organelles;
Hence, plasma membrane is the only membrane in red cell and can be easily isolated without concern about contamination by other types of membrane(Figs & 28)

29. Red Cells are biconcave in shape and lack a
nucleus and other organelles

30. Isolation of sealed and unsealed red blood cell
Cytosol (mainly hemoglobin)
is released
Isolation of sealed and unsealed red blood cell
Ghosts (plasma membrane)
Can get right-side-out and inside-out vesicles depending
on the ionic conditions of the mechanical disruption
medium

31. Sidedness of the Red Cell Membrane
Sidedness of proteins can be determined by vectorial labeling
use of a covalent reagent (radioactive or fluorescent) that binds to the portion of the protein exposed on the membrane surface
Reagent needs to be water-soluble so it won’t permeate the sealed ghost

32. Sideness of the Red Cell Membrane
For example:
label sealed right-side out vesicles
Separate proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Labeled proteins detected by their radioactivity(autoradiography) or their fluorescence (expose to UV light) (Fig )

33. SDS-PAGE separation of red blood cell proteins after
labeling of sealed
right-side-out vesicles
A, gel stained with
coomassie blue
B, drawing of the positions
of some major proteins
Glycophorin labeled
indicating it is exposed
on the outer surface of the
membrane

34. SDS-PAGE separation of red
blood cell proteins after
labeling of sealed
INSIDE-out vesicles
A, gel stained with
coomassie blue
B, drawing of the positions
of some major proteins
Glycophorin labeled
indicating it is exposed
on the INNER surface of the
membrane as well
Thus, the type of membrane
proteins can be discerned

35. Diagramatic illustration
of glycophorin in the
Red blood cell membrane
Transmembrane protein
Exposed end on the
outside surface
Exposed end on the
inner cytosolic
surface

36. Major plasma membrane proteins of the Red Blood Cell
By SDS-PAGE, has been established that there are 6 major proteins
There are many more as well, but these are the main ones (i.e. present in highest concentration)

38. Major plasma membrane proteins of the Red Blood Cell
Spectrin (peripheral protein on cytosolic surface)
Long thin flexible rod
Principal component of the cytoskelton (protein meshwork underlying surface of the red cell)

39. Major plasma membrane proteins of the Red Blood Cell
Spectrin (peripheral protein on cytosolic surface)
Maintains structural integrity of the red cell (e.g. biconvave shape)
Necessary as red cells go through small capillaries (Fig )

40. Heterodimer of 2 antiparallel polypeptide
chains, termed α and β
Heterodimers self-associate, head-
to- head, at the phosphorylated end
to form tetramers
Electron micrographs of isolated
spectrin

41. Major plasma membrane proteins of the Red Blood Cell
Spectrin (peripheral protein on cytosolic surface)
Tetramers linked together (& to band 4.1 protein) by actin filaments

43. Major plasma membrane proteins of the Red Blood Cell
Spectrin (peripheral protein on cytosolic surface)
Tetramers linked together (& to band 4.1 protein) by actin filaments
This forms a junctional complex
Serves as deformable protein mesh, allowing red cell to withstand stress on its membrane as it is forced through small capillaries (Fig 10-31)