I MEMBRANE BIOCHEMISTRY 1 4 Biological Membranes 1

I. MEMBRANE BIOCHEMISTRY § 1. 4 Biological Membranes § 1. 4 a Macromolecular Forces § 1. 4 b Artificial Membranes § 1. 4 c Lipid Bilayers

§ 1. 4 a Macromolecular Forces

Synopsis 1. 4 a - 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)

Atomic Distances - In biochemistry, the atomic distances are usually expressed in the units of Ångstrom (Å): 1 Å = 10 -10 m - In their native folded conformations, macromolecules typically measure between 10Å to 100Å in diameter - Show that there are 10Å in 1 nm: 1 nm = 10 -9 m = (10/10)*10 -9 m = (10)*10 -10 m => 10Å - Show that there are 100 pm in 1Å: 1Å = 10 -10 m = (100/100)*10 -10 m = (100)*10 -12 m => 100 pm QED Quite Easily Done! Quod Erat Demonstrandum! (What was to be demonstrated!) Anders Ångström (1814 -1874)

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

Van der Waals Forces: Overview dipole Johannes van der Waals (1837 -1923) - 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”

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

Van der Waals Forces: Three Major Types Van der Waals forces are dipole-dipole interactions that can be divided into three major categories: polar (a) Dipole-dipole interactions—interactions between permanent dipoles such as a C=O group (H-bonding is a special case of such dipolar interactions) (b) Dipole-induced-dipole interactions— permanent dipoles in groups such as – C=O can also induce a dipole moment in a neighboring group (eg -CH 3) by virtue of their ability to distort the distribution of its electronic cloud (c) London dispersion forces—these arise from the fact that the electronic cloud of nonpolar groups such as –CH 3 is not “static” but rather experiences rapidly fluctuating motions and, in so doing, generates a small transient dipole polar nonpolar

Structure of Water: van der Waals Envelope Johannes van der Waals (1837 -1923) - In chemical terms, water is dihydrogen monoxide (H 2 O)—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 2 r—ie the distance from the center of one molecule to the center of the other!

Structure of Water: sp 3 Orbitals 2 P x y z 4 x SP 3 hybridized orbitals 2 S 1 S H H O H 2 O Electronic Shell Configurations H 1 S 1 O 1 S 2. 2 S 22 p 4 Triangular face (x 4) Tetrahedron Convex vertex (x 4) Straight edge (x 6) - Water is comprised of four sp 3 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 sp 3 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

Structure of Water: Ice crystals Oxygen atom Hydrogen atom ----- H-bond - 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 => 0. 5 -1. 4 1. 0 => 0. 95 -1. 04 1. 00 => 0. 995 -1. 004

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 (1 ps = 10 -12 s) - 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 highlyordered rows of people (ice) versus a random crowd (water)

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 Na. Cl—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

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 (NH 3+)

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

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

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

Exercise 1. 4 a - 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?

§ 1. 4 b Artificial Membranes

Synopsis 1. 4 b - 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

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? !

Lipids: Artificial Membrane Systems - In amphiphiles such as fatty acids, the polar head interacts with water via Hbonding 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: (1) Micelles (monolayers) (2) Bicelles (bilayers) (3) Liposomes (bilayers) (4) Nanodiscs (bilayers) - Formation of such artificial membrane systems is driven by the hydrophobic effect–ie the ability to exclude water from their apolar tails!

(1) Detergent Micelles (Monolayers) Polar Head Central Cavity 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 higherorder 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 (3 D model)

(2) Lipid Bicelles (Bilayers) DHPC = Dihexanoylphosphatidylcholine (6: 0) DMPC = Dimyristoylphosphatidylcholine (14: 0) Polar Head Longertail DMPC Shortertail DHPC Water Apolar Tails Phospholipid (amphiphile) Bicelle Disk-like (3 D model) 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 (!)

(3) Liposomes (Bilayers) Central Cavity Lipid Nutrient Bilayer Deliverywaves with - When disrupted by physical treatments such as sonication (agitation with ultrasonic ~ mm), phospholipid bicelles (lipid bilayers) form liposomes— 3 D 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

(4) Nanodiscs (Bilayers) Membrane scaffold proteins (MSPs) Lipid - Nanodiscs are comprised of a central phospholipid bilayer wherein the outer bilayer 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 3 D architectures)

Exercise 1. 4 b - 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?

§ 1. 4 c Lipid Bilayers

Synopsis 1. 4 c - 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”

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

Lipid Bilayer Is a 2 D Fluid Lateral Diffusion Transverse Diffusion (Flipflop) - 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 (2 D) fluid—cf the mobility of terrestrial animals (2 D) vs birds/fish (3 D)!

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

Effect of Cholesterol on Phase Transition 50 40 30 Cholesterol / % 20 Heat Absorbed 10 0 Cholesterol Phospholipids T / C - 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

Lipid Bilayer Harbors Membrane Proteins Peripheral Membrane Protein Integral Membrane Proteins Extracellula r Lipid Bilaye r Peripheral Membrane Protein monotopic polytopic Cytoplasmi c - 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—

Integral Membrane Proteins (IMPs) Extracellula r Bacteriorhodopsin (polytopic -helical) Omp. F (antiparallel -barrel) Lipid Bilaye r Cytoplasmi c - 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

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

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 2 D 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!

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

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): 1) Flippases—catalyze the movement of specific phospholipids from the extracellular face to the cytosolic leaflet in an ATP-dependent manner 2) Floppases—catalyze the movement of specific phospholipids from the cytoplasmic face to the extracellular leaflet in an ATP-dependent manner 3) 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

Exercise 1. 4 c - 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