1. I. MEMBRANE BIOCHEMISTRY
§1.4 Biological Membranes
§1.4a Macromolecular Forces
§1.4b Artificial Membranes
§1.4c Lipid Bilayers

2. §1.4a Macromolecular Forces

3. Synopsis 1.4a
In order to understand biochemical phenomena such as the assembly of biological membranes, folding of proteins, ligand binding to proteins, and substrate binding to enzymes, one must understand the nature of intermolecular forces acting on biological molecules
Such Intermolecular forces can be broadly be classified as:
(1) ionic interactions
(2) van der Waals forces (eg hydrogen bonding, dipolar interactions)
The exclusion of nonpolar groups from polar surroundings so as to maximize the entropy of water molecules is called the “hydrophobic effect”
Atomic distances are measured in the units of Ångström (Å)

4. Atomic Distances
Anders Ångström
( )
In biochemistry, the atomic distances are usually expressed in the units of Ångstrom (Å):
1 Å = m
In their native folded conformations, macromolecules typically measure between 10Å to 100Å in diameter
Show that there are 10Å in 1nm:
1nm = 10-9m = (10/10)*10-9m = (10)*10-10m => 10Å
Show that there are 100pm in 1Å:
1Å = 10-10m = (100/100)*10-10m = (100)*10-12m => 100pm
QED
Quite Easily Done!
Quod Erat Demonstrandum!
(What was to be demonstrated!)

5. Typical Bond Energies in Biomolecules
Non-covalent forces underlying intermolecular interactions between biological molecules (or biomolecules) can be divided into TWO major categories:
(1) Ionic interactions —eg between oppositely charged ions such as Na+ and Cl-
(2) van der Waals forces —interactions due to dipoles
Although relatively weak, the London dispersion forces are nonetheless important—the major reason for the liquidity of non-polar substances such as benzene

6. Van der Waals Forces: Overview
Johannes van der Waals
( )
dipole
The tendency of an atom to attract a shared pair of electrons (eg within a covalent bond) toward itself results from a chemical property termed “elecronegativity”
Due to such electronegativity, electrons involved in mediating covalent bonds between a pair of atoms are often not equally distributed but rather become slightly pulled toward one or the other bonding partner, thereby resulting in the formation of diploes (or charge separation)—this phenomenon is called “electrostatic polarization”
Under such electrostatic polarization in the context of a covalent bond between a pair of atoms, one atom carries a slightly negative charge (-) while the other a slightly positive charge (+)
Intermolecular Interactions between oppositely charged ends of dipoles (dipole-dipole interactions) are broadly known as “van der Waals forces”

7. Van der Waals Forces: Hydrogen Bonding+ -
If dipole-dipole interactions occur between an electropositive H atom bonded to another highly electronegative atom (such as O or N), the resulting van der Waals forces are called “hydrogen bonding”—ie hydrogen bonding (or H-bonding) is a special case of van der Waals forces due to its rather strong nature coupled with its ubiquity in biological systems
Hydrogen bonding—represented by a dotted or dashed line—is the supreme attractive force that renders water a liquid at room temperature
Changes in H-bonding pattern impart upon water the ability to exist in a liquid or crystalline (ice) form
Because of charge separation or polarization of electronic clouds of H and O atoms, water is described as being a highly “polar” molecule—such polarity of water enables it to act both as H-bond donor as well as an H-bond acceptor in biochemical processes

8. Van der Waals Forces: Three Major Types
polar
Van der Waals forces are dipole-dipole interactions that can be divided into three major categories:
Dipole-dipole interactions—interactions between permanent dipoles such as a -C=O group (H-bonding is a special case of such dipolar interactions)
Dipole-induced-dipole interactions—permanent dipoles in groups such as –C=O can also induce a dipole moment in a neighboring group (eg -CH3) by virtue of their ability to distort the distribution of its electronic cloud
London dispersion forces—these arise from the fact that the electronic cloud of nonpolar groups such as –CH3 is not “static” but rather experiences rapidly fluctuating motions and, in so doing, generates a small transient dipole
polar
nonpolar
nonpolar

9. Structure of Water: van der Waals Envelope
Johannes van der Waals
( )
In chemical terms, water is dihydrogen monoxide (H2O)—wherein two hydrogen atoms are covalently bonded to an oxygen atom
While essential to life, dihydrogen monoxide is a lethal chemical (!) in that it can rapidly corrode and destroy most materials!
van der Waals envelope (or surface) is the approximate perimeter of a molecule as demarcated by the outer boundary of the surrounding cloud of electrons—the distance from the center of the molecule to the van der Waals envelope is called the “van der Waals radius”
van der Waals radius (r) of water is 1.4Å—two water molecules cannot get closer to each other more than 2r—ie the distance from the center of one molecule to the center of the other!

10. Structure of Water: sp3 Orbitals
Electronic Shell Configurations
H  1S1
O  1S2.2S22p4 1S 2S 2P H O x y z
4 x SP3
hybridized
orbitals
H2O
Tetrahedron
Triangular face (x4)
Convex vertex (x4)
Straight edge (x6) 
Water is comprised of four sp3 hybridized (or mixed) orbitals—two of which are associated with H atoms, while the other two arise from the two non-bonding pairs of electrons
The four sp3 orbitals of water adopt a tetrahedral geometry—ie each orbital occupies one of the four corners or vertices (singular  vertex) in a tetrahedral arrangement:
if  < 180 => convex vertex
if  > 180 => concave vertex

11. Structure of Water: Ice crystals
----- H-bond
Oxygen atom
Hydrogen atom
Ice is a crystal of an highly ordered network of hydrogen-bonded water molecules
In ice, each water molecule interacts tetrahedrally with four other neighboring (or surrounding) water molecules
In ice, H-bonds are highly stable (static)
Because of a regular “open” ordered network of hydrogen bonding, water expands on freezing—ie ice (0.92 g/ml) has a lower density than liquid water (1.00 g/ml)
What is the difference between 1, 1.0, and 1.00?!
=>
=>
1.00 =>

12. Structure of Water: Liquid
3-mer
4-mer
5-mer
Liquid water consists of a rather “loose” network of hydrogen-bonded water molecules—ie water molecules rapidly fluctuate and tumble on a picosecond (ps) timescale (1ps = 10-12s)
Unlike ice, liquid water thus harbors an highly disordered and irregular structure
In liquid water, H-bonds are highly unstable (dynamic)
Nevertheless, water molecules transiently engage in rings of three (3-mer), four (4-mer), or five (5-mer) molecules in liquid
Because of their irregularity in liquid, water molecules can pack together much more tightly than in ice, thereby rendering water (1.00 g/ml) more dense than ice (0.92 g/ml)—cf highly-ordered rows of people (ice) versus a random crowd (water)

13. Hydration: Solvation of Ionic Substances
Cations are shielded by
electronegative O atoms
Anions are shielded by
electropositive H atoms
Water is often described as a “universal solvent” due to the fact that its polar character renders it an excellent solvent for hydrophilic substances—eg those with polar or ionic character
The ability of water to dissolve polar substances—such as NaCl—arises from the fact that its dipolar character enables it to weaken attractive forces between oppositely charged Na+ and Cl- ions
Multiple water molecules can surround each ion and neutralize its charge in a phenomenon referred to as “hydration”—or solvation in generic terms

14. Hydration: Solvation of Polar Substances
Hydroxyl
Keto
Carboxylate
Amino
Water is also an excellent solvent for polar substances for the same reason that it is for ionic substances
The dipolar character of water enables it to engage in H-bonding with other polar groups such as hydroxyl (OH), carboxylate (O=CO-), keto (C=O) and amino (NH3+)

15. Hydrophobic Effect: An Entropic Phenomenon
Apolar (or nonpolar) molecules such as oils and lipids aggregate when in contact with water—ie they tend to “stick” together rather than dissolve in water—why?!
Such ability of apolar molecules to minimize contact with water or vice versa is termed the “hydrophobic effect”—what thermodynamic force drives the hydrophobic effect? Enthalpic (H) or entropic (TS)?
H accompanying the transfer of apolar substances from water to an apolar solvent is unfavorable (H >= 0), while TS is consistently favorable (TS > 0)
Thus, the hydrophobic effect is largely driven by an entropic force in that the ability of apolar substances to aggregate confers upon surrounding water molecules an entropic advantage—ie exclusion of water molecules enables them to move and tumble freely in lieu of being “locked” or “entrapped” in an ordered manner with apolar neighbors

16. Hydrophobic Effect: Orientation of Water Molecules
Maintenance of intramolecular
H-bonding network is critical to the
random motion of water molecules
Intrusion of apolar solute into water
disrupts such extensive network due
to its inability to engage in H-bonding
interactions
Accordingly, water molecules orient
away from the surface of the apolar
solute to engage in H-bonding network
with bulk water molecules—the surrounding
water molecules that are not in direct contact
with the solute
Such orientation constitutes an ordering of the water structure (as their degree of freedom or the number of ways in which they can hydrogen bond becomes restricted)
In order to minimize such ordering of water molecules, apolar molecules aggregate so as to minimize their surface area in contact with water and thereby maximize the overall entropy of the system

17. Hydrophobic Effect: Aggregation of Apolar Substances
Individual hydration of
apolar substances increases their surface area in contact with water—thereby resulting in greater loss of entropy
Aggregation of apolar substances minimizes their surface area in contact with water—thereby resulting in lesser loss of entropy
Hydrophobic effect is central to many (bio)physicochemical phenomena:
Separation of oil and water
Membrane bilayer integrity
Folding of proteins in water and lipid bilayer

18. Exercise 1.4a
Sketch a diagram of a water molecule and indicate the ends that bear partial positive and negative charges
Compare the structures of ice and water with respect to the number and geometry of hydrogen bonds
Describe the nature and relative strength of covalent bonds, ionic interactions, and van der Waals forces
Explain why polar substances dissolve in water while nonpolar substances do not
What is the role of entropy in the hydrophobic effect?

19. §1.4b Artificial Membranes

20. Synopsis 1.4b
In water, apolar molecules such as fats and oils (eg triglycerides) aggregate—ie they form non-homogeneous droplets—due to the hydrophobic effect
In contrast, amphiphilic molecules (eg detergents and phospholipids) associate (but do not solubilize or dissolve!) into monolayers (micelles) or bilayers (bicelles) so as to form a homogenous solution with water
The ability of amphiphilic substances to form micelles or bicelles so as to shield their hydrophobic groups while exposing their hydrophilic groups to water is driven by the hydrophobic effect

21. Amphiphiles: Fatty Acids and Detergents
Sodium Dodecyl Sulfate (SDS)
Apolar Tail
Polar Head
Most biomolecules harbor both hydrophilic and hydrophobic characters—ie they possess polar (eg polarized and/or charged) and apolar regions/segments
Such biomolecules with hybrid character—such as fatty acids (eg palmitate) and detergents (eg SDS)—are said to be “amphiphilic” or “amphipathic”:
amphi  both
philia  attraction/liking
phobia  disliking/repulsion
pathos  to suffer/harbor
Detergents are used as cleaning agents due to their ability to interact with both water (through their charged head) and oily substances or stains (through their nonpolar tail)
Do amphiphiles such as fatty acids and detergents aggregate or solubilize in water?!

22. Lipids: Artificial Membrane Systems
In amphiphiles such as fatty acids, the polar head interacts with water via H-bonding while the apolar tails exclude water on thermodynamic grounds
Accordingly, amphiphiles aggregate in water in an highly ordered manner
Such ordered aggregates of amphiphiles form four distinct type of artificial/synthetic membrane systems:
Micelles (monolayers)
Bicelles (bilayers)
Liposomes (bilayers)
Nanodiscs (bilayers)
Formation of such artificial membrane systems is driven by the hydrophobic effect–ie the ability to exclude water from their apolar tails!

23. (1) Detergent Micelles (Monolayers)
Central Cavity
Polar Head
Water
Apolar Tail
SDS (detergent)
SDS (amphiphile)
Spherical Micelle
(monolayer)
In aqueous solution, single-tailed detergents (amphiphilic) such as sodium dodecyl sulfate (SDS) and fatty acids associate (or aggregate) into higher- order structures called micelles—m for monolayer!
Such arrangement allows the non-polar (apolar) tails to avoid contact with water while allowing their polar head groups to interact with the solvent
Formation of such micelles is driven by the fact that it is thermodynamically more favorable for the non-polar detergent tails to exclude water and engage in van der Waals contacts with the neighboring tails in addition to being entropically favorable (TS > 0)
The central cavity may also become filled with water depending on the concentration of detergent molecules
Spherical Micelle
(3D model)

24. Phospholipid (amphiphile) Disk-like Bicelle (bilayer)
(2) Lipid Bicelles (Bilayers)
DHPC = Dihexanoylphosphatidylcholine (6:0)
DMPC = Dimyristoylphosphatidylcholine (14:0)
Longer-tail DMPC
Shorter-tail DHPC
Polar Head
Water
Apolar Tails
Phospholipid (amphiphile)
Bicelle
(3D model)
Disk-like Bicelle (bilayer)
In a manner akin to the formation of detergent micelles, double-tailed amphiphilic molecules such as phospholipids associate (or aggregate) into higher-order disc-like structures called bicelles—b for bilayer!
Unlike less crowded single-tailed detergents, unfavorable steric clashes between double-tailed lipids require them to pack into what are essentially disk-like bilayers (bicelles) in lieu of monolayers (micelles)—albeit with monolayers at the edges!
Within such a bicelle, lipid heads stick out on the surface on each side, while tails align up in a more or less extended conformation against each other internally to generate what is predominantly a “lipid bilayer”—this is the physicochemical basis of the assembly of biological membranes!
The ability of such amphiphilic molecules to exclude water from their non-polar surfaces is of course (!) called the “hydrophobic effect”—it is also the basis of folding of proteins into 3D shapes!

25. (3) Liposomes (Bilayers)
Central
Cavity
Lipid Bilayer
Nutrient Delivery
When disrupted by physical treatments such as sonication (agitation with ultrasonic waves with  ~ mm), phospholipid bicelles (lipid bilayers) form liposomes—3D spherical structures fully enclosed by a single lipid bilayer with a central aqueous cavity
Such liposomes not only serve as artificial membranes for research but can also be used as a vehicle for the delivery of hydrophilic nutrients and drugs (encapsulated in the central cavity) that do not readily diffuse through the cell membranes
Conversely, hydrophobic nutrients and drugs can also be delivered to specific tissues by virtue of the fact that they readily dissolve into the lipid bilayer of the liposomes
Upon reaching their target site, liposomes fuse with biological membranes, thereby emptying their contents into the inside of the cell

26. Membrane scaffold proteins
(4) Nanodiscs (Bilayers)
Lipid bilayer
Membrane scaffold proteins
(MSPs)
Nanodiscs are comprised of a central phospholipid bilayer wherein the outer boundary/perimeter of apolar tails is shielded by amphipathic proteins called “membrane scaffold proteins (MSPs)” in a double-belt fashion
MSPs are usually modified apolipoproteins—proteins involved in lipid metabolism—that harbor amphipathic character
Nanodiscs serve as excellent artificial membranes for research in that they represent a more native system for the stabilization/folding of membrane proteins than lipsomes, bicelles and micelles
Nanodiscs may look like lipid bicelles (!)—but nanodsics differ from bicelles in that while the former represent relatively homogeneous structures, the latter are highly heterogenous (poorly defined 3D architectures)

27. Exercise 1.4b Describe the four types of artificial membranes
Why do phospholipids form bilayers rather than monolayers?
How do liposomes differ from micelles?
How do nanodiscs differ from bicelles?

28. §1.4c Lipid Bilayers

29. Synopsis 1.4c
The cell membranes of living organisms, including intracellular organelles such as the mitochondrion and nucleus, are composed of lipid bilayers
Within such a lipid bilayer, the tails of lipids align up against each other while the heads point either toward the exterior or the interior of the cell in a manner akin to a liposome architecture
In addition to lipids, a wide plethora of so-called integral membrane proteins (IMPs) penetrate the lipid bilayer and laterally “swim” along the plane of the bilayer
IMPs contain a transmembrane structure consisting of α-helices or a -barrel with a hydrophobic surface
The dynamic arrangement and interactions of membrane lipids and proteins are described by the “fluid mosaic model”

30. Cell Membrane is a Lipid Bilayer
All living cells are enclosed by a “semi-permeable” barrier or cell membrane called “lipid bilayer”
The membranes of intracellular organelles such as the mitochondrion and nucleus are also composed of a lipid bilayer

31. Transverse Diffusion (Flip-flop)
Lipid Bilayer Is a 2D Fluid
Lateral Diffusion
Transverse Diffusion (Flip-flop)
Owing to the ability of lipids to rapidly exchange with each other within the plane of the same bilayer leaflet (lateral diffusion), biological membranes are highly dynamic and fluid structures
On the other hand, exchange of lipids across the bilayer leaflets (transverse diffusion) is extremely rare due to thermodynamic constraints—such flip-flop requires the outer polar head to rotate and become momentarily immersed in the apolar environment of lipid tails at the core of the bilayer
The lipid tails within the core of the bilayer are under constant motion due to the free rotation about the C-C bond, while the motion of head groups is relatively restricted due to steric clashes or unfavorable polarity
Because of the mobility of lipids primarily within the plane of the same bilayer leaflet, the lipid bilayer is often described as a two-dimensional (2D) fluid—cf the mobility of terrestrial animals (2D) vs birds/fish (3D)!

32. Lipid Bilayer Experiences Phase Transition
Liquid Solution
Viscous fluid with high mobility (T > Tm)
Liquid Crystal
Gel-like solid with an ordered array (T < Tm)
Although a pre-requisite for the living cell, the fluidity of lipid bilayer is in general highly dependent upon temperature (T) and lipid composition
Lipid bilayer undergoes a dramatic phase transition in vitro from being a highly viscous fluid (liquid solution) to a gel-like solid (liquid crystal) as the temperature is lowered to and below a certain threshold value (usually around 25C)—this is called transition temperature (Tm)—the temperature at which the bilayer “melts” (cf water-ice phase transition)!
Tm is highly dependent upon both the chain length (x) and degree of saturation (m) of fatty acids of membrane lipids—longer the chain length and/or higher the degree of saturation, higher the Tm!
Changing the composition of fatty acids of membrane lipids can attune the Tm of lipid bilayer (so as to maintain its fluidity at ambient temperature)—a necessity for cold-blooded animals such as fish
Fortunately, biological membranes experience little or negligible aforementioned bilayer phase transition—how so? Enter cholesterol!

33. Effect of Cholesterol on Phase Transition10 20 30 40 50
T / C
Heat Absorbed
Cholesterol / %(mol/mol)
Cholesterol
Phospholipids
By virtue of its ability to snug in between phospholipids, cholesterol serves as a “fluidity buffer” and either broadens or completely abolishes the phase transition observed in lipid bilayers in vitro in response to changes in temperature!
When T > Tm, cholesterol (due to its highly rigid fused ring system) decreases bilayer fluidity by interfering with the motions of lipid tails
When T < Tm, cholesterol increases bilayer fluidity by disrupting close packing of lipid tails

34. Integral Membrane Proteins
Lipid Bilayer Harbors Membrane Proteins
Integral Membrane Proteins
Extracellular
Peripheral Membrane Protein
polytopic
monotopic
Lipid Bilayer
Peripheral Membrane Protein
Cytoplasmic
In addition to lipids, proteins also constitute a major component of biological membranes— such proteins are referred to as “membrane proteins”
Membrane proteins are classified into “integral” or “peripheral” depending on the nature of their interactions with the lipid bilayer
Integral membrane proteins (IMPs) traverse through the lipid bilayer once (monotopic) or multiple times (polytopic)—IMPs are highly hydrophobic and insoluble (precipitate out) in water
Peripheral membrane proteins (PMPs) adhere to the surface of either the inner or outer leaflet of the bilayer via association with lipid head groups or non-transmembrane regions of IMPs— PMPs are water-soluble

35. (polytopic -helical) (antiparallel -barrel)
Integral Membrane Proteins (IMPs)
Bacteriorhodopsin
(polytopic -helical)
OmpF
(antiparallel -barrel)
Extracellular
Lipid Bilayer
Cytoplasmic
IMPs are essentially amphiphiles—the protein regions (or transmembrane segments) immersed within the milieu of the bilayer are predominantly composed of non-polar amino acid residues, while intervening regions (loops) residing on the extracellular and cytoplasmic faces are by and large dominated by polar and charged residues
Transmembrane segments traversing lipid bilayers (biological membranes) usually adopt two major folds or topologies—-helical or -barrel
-barrel transmembrane proteins are essentially comprised of a large multi-stranded -sheet that twists and coils to form a closed hollow channel—so as to allow passive diffusion of nutrients, salts and water
-helical transmembrane proteins can either traverse through the bilayer once (monotopic) or multiple times (polytopic) to form the so-called helical bundle—and they conduct a multitude of roles from signal transduction (cell surface receptors) to energy generation (proton pumps)

36. Peripheral Membrane Proteins (PMPs)
PMPs adhere to the surface of either the inner or outer leaflet of the bilayer via association with lipid head groups or non-transmembrane regions of IMPs
PMPs are essentially “water-soluble” proteins that impart structural and functional versatility upon biological membranes—eg attachment of spectrin, actin and ankyrin to the inner leaflet of biological membranes not only serves as the “membrane skeleton” that gives the cell shape but also provides a framework for the smooth integration and operation of signaling networks running from the nucleus to the events occurring at the cell surface

37. Fluid Mosaic Model
Lipid bilayer can be described as a “mosaic”—a composite structure made up of a heterogeneous mixture of constituent components such as lipids and proteins arranged in an “orderly” manner—the latter are further decorated with oligosaccharides to form what are called “glycoproteins” or “proteoglycans”
In addition to the ability of lipids to freely exchange within the same leaflet of lipid bilayer, integral membrane proteins also undergo a similar lateral diffusion along the plane of the bilayer—they essentially “float” in a “hydrophobic sea” of lipid bilayer!
Given such 2D fluid of the bilayer with lipids and proteins constantly on the move, the lipid bilayer is best envisioned as a “fluid mosaic”—such a model accounts for most of its biological properties!

38. Asymmetric Distribution of Lipids
Asymmetric Distribution of Lipids in the Cell Membrane of Human Erythrocyte
Protein components of biological membranes are not evenly distributed on both sides—eg the oligosaccharide moities of glycoproteins are almost exclusively attached to their extracellular regions (where they play a central role in mediating cell-cell interactions)
In a similar manner, lipids are also asymmetrically distributed on each face of the lipid bilayer—eg sphingomyelin and phosphatidylcholine are predominantly located in the extracellular leaflet of erythrocytes, whereas phosphatidylethanolamine and phosphaitdylserine are on the cytoplasmic face
Such asymmetric distribution of lipids is necessary to attune specific cell types for their specific needs and physiological functions

39. Transbilayer Movement of Lipids
1) Flippases
2) Floppases
3) Scramblases
extracellular
Transverse
Diffusion
Since lipids rarely flip across a bilayer spontaneously by transverse diffusion, asymmetric distribution of phospholipids across biological membranes is achieved via three transmembrane transporters (sphingolipids are not flipped):
Flippases—catalyze the movement of specific phospholipids from the extracellular face to the cytosolic leaflet in an ATP-dependent manner
Floppases—catalyze the movement of specific phospholipids from the cytoplasmic face to the extracellular leaflet in an ATP-dependent manner
Scramblases—aid redistribution or equilibration of phospholipids by virtue of their ability to translocate phospholipids between bilayer leaflets in a bidirectional manner without the need for ATP-coupled hydrolysis

40. Exercise 1.4c
Why do glycerophospholipids and sphingolipids—but not fatty acids—form bilayers?
Explain why lateral diffusion of membrane lipids is faster than transverse diffusion
What factors influence the fluidity of a bilayer?
What are the two types of secondary structures that occur in transmembrane proteins?
Describe the fluid mosaic model