Biochemistry, 4th Edition P29 pdf

The first, called peripheral pro-teins or extrinsic proteins, includes those that do not penetrate the bilayer to any significant degree and are associated with the membrane by virtue of i

or in the membrane and the transport of ions, sugars, and amino acids across

mem-branes; and organizes and directs hundreds of cell signaling events

The Composition of Membranes Suits Their Functions

Biological membranes may contain as much as 75% to 80% protein (and only 20%

to 25% lipid) or as little as 15% to 20% protein Membranes that carry out many

enzyme-catalyzed reactions and transport activities (the inner mitochondrial

mem-brane, chloroplast membranes, and the plasma membrane of Escherichia coli, for

example) are typically richer in protein, whereas membranes that carry out fewer

protein-related functions (myelin sheaths, the protective coating around neurons,

for example) are richer in lipid

Cellular mechanisms adjust lipid composition to functional needs Thus, for

ex-ample, the lipid makeup of red blood cell membranes is consistent across species,

whereas the lipid complement of different (specialized) membranes within a

par-ticular cell type (rat liver, Figure 9.2) reflects differences of function Plasma

mem-branes are enriched in cholesterol but do not contain diphosphatidylglycerol

(a)

FIGURE 9.1 Electron micrographs of several different membrane structures: (a) Plasma membrane of

Menoid-ium, a protozoan (b) Two plasma membranes from adjacent neurons in the central nervous system (c) Golgi

apparatus (d) Many membrane structures are evident in pancreatic acinar cells.

T V

Nuclear membrane

Plasma membrane Lysosomes Mitochondria

Phosphatidylcholine

Phosphatidylethanolamine

Sphingolipids

Phosphatidylinositol

Phosphatidylserine

Cardiolipin

Minor lipids

Cholesterol

Golgi apparatus

FIGURE 9.2 The lipid composition of rat liver cell membranes, in weight percent (Adapted from Andreoli, T E., 1987.

Membrane Physiology, 2nd ed Chapter 27, Table II, and Daum, G., 1985 Lipids of mitochondria Biochimica et Biophysica Acta

(cardiolipin), whereas mitochondria contain considerable amounts of cardiolipin (essential for some mitochondrial proteins) and no cholesterol The protein com-ponents of membranes vary even more greatly than their lipid compositions

Lipids Form Ordered Structures Spontaneously in Water Monolayers and Micelles Amphipathic lipids spontaneously form a variety of struc-tures when added to aqueous solution All these strucstruc-tures form in ways that minimize contact between the hydrophobic lipid chains and the aqueous milieu For example, when small amounts of a fatty acid are added to an aqueous solution, a monolayer is formed at the air–water interface, with the polar head groups in contact with the wa-ter surface and the hydrophobic tails in contact with the air (Figure 9.3) Few lipid molecules are found as monomers in solution

Further addition of fatty acid eventually results in the formation of micelles

Micellesformed from an amphipathic lipid in water position the hydrophobic tails

in the center of the lipid aggregation with the polar head groups facing outward

Amphipathic molecules that form micelles are characterized by a unique critical micelle concentration, or CMC Below the CMC, individual lipid molecules

pre-dominate Nearly all the lipid added above the CMC, however, spontaneously forms micelles Micelles are the preferred form of aggregation in water for detergents and soaps Some typical CMC values are listed in Figure 9.4

Lipid Bilayers Lipid bilayersconsist of back-to-back arrangements of monolayers (Figure 9.3) The nonpolar portions of the lipids face the middle of the bilayer, with the polar head groups arrayed on the bilayer surface Phospholipid bilayers form

(e)

Air

Water Monolayer

Bilayer

Normal

(d) Multilamellar vesicle

(b) Micelles

(a) Monolayers and bilayers (c) Unilamellar vesicle

Inside-out

Water

FIGURE 9.3 Several spontaneously formed lipid structures Drawings of (a) monolayers and bilayers, (b) mi-celles, (c) a unilamellar vesicle, (d) a multilamellar vesicle, and (e) an electron micrograph of a multilamellar

Golgi structure.

rapidly and spontaneously when phospholipids are added to water, and they are

sta-ble structures in aqueous solution As opposed to micelles, which are small,

self-limiting structures of a few hundred molecules, bilayers may form spontaneously over

large areas (108nm2or more) Because exposure of the edges of the bilayer to solvent

is highly unfavorable, extensive bilayers normally wrap around themselves and form

closed vesicles (Figure 9.3) The nature and integrity of these vesicle structures are

very much dependent on the lipid composition Phospholipids can form either

uni-lamellar vesicles (with a single lipid bilayer), known as liposomes, or multiuni-lamellar vesicles.

These latter structures are reminiscent of the layered structure of onions

Liposomesare highly stable structures, a consequence of the amphipathic

na-ture of the phospholipid molecule Ionic interactions between the polar head

groups and water are maximized, whereas hydrophobic interactions (see Chapter

2) facilitate the association of hydrocarbon chains in the interior of the bilayer

The formation of vesicles results in a favorable increase in the entropy of the

solu-tion, because the water molecules are not required to order themselves around the

lipid chains It is important to consider for a moment the physical properties of the

bilayer membrane, which is the basis of vesicles and also of natural membranes

Bi-layers have a polar surface and a nonpolar core This hydrophobic core provides a

substantial barrier to ions and other polar entities The rates of movement of such

species across membranes are thus quite slow However, this same core also

pro-vides a favorable environment for nonpolar molecules and hydrophobic proteins

We will encounter numerous cases of hydrophobic molecules that interact with

membranes and regulate biological functions in some way by binding to or

em-bedding themselves in membranes

The Fluid Mosaic Model Describes Membrane Dynamics

In 1972, S J Singer and G L Nicolson proposed the fluid mosaic model for

mem-brane structure, which suggested that memmem-branes are dynamic structures

com-posed of proteins and phospholipids In this model, the phospholipid bilayer is a

fluid matrix, in essence, a two-dimensional solvent for proteins Both lipids and

pro-teins are capable of rotational and lateral movement

Singer and Nicolson also pointed out that proteins can be associated with the

surface of this bilayer or embedded in the bilayer to varying degrees (Figure 9.5)

Triton X-100

CH3

CH3

10

Mr CMC Micelle Mr

625 0.24 mM 90–95,000

Octyl glucoside

H

HO

OH H

H

CH2OH

O

H

(CH2)

C 12 E 8 (Dodecyl octaoxyethylene ether)

(OCH2CH2)

8

Structure

538

O

CH2

CH3

CH3

OH

CH3

OH

FIGURE 9.4 The structures of some common detergents and their physical properties Micelles formed by

detergents can be quite large Triton X-100, for example, typically forms micelles with a total molecular mass of

90 to 95 kD This corresponds to approximately 150 molecules of Triton X-100 per micelle.

They defined two classes of membrane proteins The first, called peripheral pro-teins (or extrinsic proteins), includes those that do not penetrate the bilayer to any

significant degree and are associated with the membrane by virtue of ionic interac-tions and hydrogen bonds between the membrane surface and the surface of the protein Peripheral proteins can be dissociated from the membrane by treatment with salt solutions or by changes in pH (treatments that disrupt hydrogen bonds

and ionic interactions) Integral proteins (or intrinsic proteins), in contrast, possess

hydrophobic surfaces that can readily penetrate the lipid bilayer itself, as well as sur-faces that prefer contact with the aqueous medium These proteins can either insert into the membrane or extend all the way across the membrane and expose them-selves to the aqueous solvent on both sides Singer and Nicolson also suggested that

a portion of the bilayer lipid interacts in specific ways with integral membrane pro-teins and that these interactions might be important for the function of certain membrane proteins Because of these intimate associations with membrane lipid, integral proteins can be removed from the membrane only by agents capable of breaking up the hydrophobic interactions within the lipid bilayer itself (such as detergents and organic solvents) The fluid mosaic model became the paradigm for modern studies that have advanced our understanding of membrane structure and function

The Thickness of a Membrane Depends on Its Components Electron micro-graphs of typical cellular membranes show the thickness of the entire membrane— including lipid bilayer and embedded protein—to be 50 Å or more Electron mi-croscopy, NMR, and X-ray and neutron diffraction measurements have shown that membrane thickness is influenced by the particular lipids and proteins in the mem-brane The thickness of a phospholipid bilayer made from dipalmitoyl phos-phatidylcholine, measured as the phosphorus-to-phosphorus spacing, is about 37 Å, and the hydrophobic phase of such membranes is approximately 26 Å thick Nat-ural membranes are thicker overall than simple lipid bilayers because many mem-brane proteins extend out of the bilayer significantly

Among the known membrane protein structures, there is considerable variation

in the hydrophobic surface perpendicular to the membrane plane If the hy-drophobic surface of the protein is larger or smaller than the lipid bilayer, the thick-ness of the lipid bilayer must be increased or decreased The change in bilayer thickness due to membrane proteins can be as much as 5 Å

Integral proteins

Peripheral protein

Phospholipid membrane

Glycolipid Oligosaccharide side chain

Cholesterol

FIGURE 9.5 The fluid mosaic model of membrane structure proposed by S J Singer and G L Nicolson In this model, the lipids and proteins are assumed to be mobile; they can diffuse laterally in the plane of the mem-brane Transverse motion may also occur, but it is much slower.

Lipid Chains May Bend and Tilt in the Membrane The long hydrocarbon chains

of lipids are typically portrayed as more or less perpendicular to the membrane

plane (Figure 9.3) In fact, the hydrocarbon tails of phospholipids may tilt and bend

and adopt a variety of orientations Typically, the portions of a lipid chain near the

membrane surface lie most nearly perpendicular to the membrane plane, and lipid

chain ordering decreases toward the end of the chain (toward the middle of the

bilayer)

Membranes Are Crowded with Many Different Proteins Membranes are crowded

places, with a large number of proteins either embedded or associated in some way

The E coli genome codes for more than a thousand membrane proteins Moreover,

as more membrane protein structures are determined (Figure 9.6), it has become

apparent that many membrane proteins have large structures extending outside the

lipid bilayer that share steric contacts and other interactions Donald Engelman has

suggested that most membranes are more crowded than first portrayed in Singer

and Nicolson’s model (Figure 9.7)

200

180

160

140

120

100

80 60 40 20 0

1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005

Total number of membrane protein structures solved

Year

FIGURE 9.6 Membrane protein structures, by year published (Data from the Web site Membrane Proteins of Known

3D Structure at the laboratory of Stephen White, http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html, and from the

Web site of Hartmut Michel, http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html.)

FIGURE 9.7 An updated model for membrane structure,

as proposed by Donald Engelman (Adapted from Engelman, D., 2005 Membranes are more mosaic than fluid.

9.2 What Are the Structure and Chemistry

of Membrane Proteins?

Although the lipid bilayer constitutes the fundamental structural unit of all biolog-ical membranes, proteins carry out essentially all of the active functions of mem-branes Singer and Nicolson defined peripheral proteins as globular proteins that interact with the membrane mainly through electrostatic and hydrogen-bonding interactions, and integral proteins as those that are strongly associated with the lipid

bilayer Another class of proteins not anticipated by Singer and Nicolson, the lipid-anchored proteins,is important in a variety of functions in different cells and tis-sues These proteins associate with membranes by means of a variety of covalently linked lipid anchors

Peripheral Membrane Proteins Associate Loosely with the Membrane

Peripheral proteins can bind to membranes in several ways (Figure 9.8) They may form ionic interactions and hydrogen bonds with polar head groups of membrane lipids or with other (integral) proteins, or they may interact with the nonpolar membrane core by inserting a hydrophobic loop or an amphipathic -helix

Exam-ples of each of these interaction types are shown in Figure 9.9

Integral Membrane Proteins Are Firmly Anchored in the Membrane

Hundreds of structures of integral membrane proteins are now available in the Pro-tein Data Bank, and the number of membrane proPro-tein structures is doubling about every 3 years The known structures show a surprising diversity, but in all cases the

ⴙ ⴚ ⴙ ⴚ

Association with integral protein

Hydrophobic loop

Amphipathic

-helix

Ionic and H-bond interactions

FIGURE 9.8 Four possible modes for the binding of peripheral membrane proteins.

FIGURE 9.9 Models for membrane association of peripheral proteins (a) Bee venom phospholipase A2 (pdb id

 1POC), (b) p40 phox PX domain of NADH oxidase (pdb id  1H6H), and (c) PH domain of phospholipase C

(pdb id  1MAI).

portions of the protein in contact with the nonpolar core of the lipid bilayer are

dominated by -helices or -sheets, because these secondary structures neutralize

the highly polar NOH and CPO functions of the peptide backbone through

H-bond formation

Proteins with a Single Transmembrane Segment In proteins that are anchored

by a single hydrophobic segment, that segment typically takes the form of an

-helix One of the best examples is glycophorin Most of glycophorin’s mass is

ori-ented on the outside surface of the red blood cell, exposed to the aqueous milieu

(Figure 9.10) Hydrophilic oligosaccharide units are attached to this extracellular

domain These oligosaccharide groups constitute the ABO and MN blood group

antigenic specificities of the red cell Glycophorin has a total molecular weight of

about 31,000 and is approximately 40% protein and 60% carbohydrate The

gly-cophorin primary structure consists of a segment of 19 hydrophobic amino acid

residues with a short hydrophilic sequence on one end and a longer hydrophilic

sequence on the other end The 19-residue sequence is just the right length to span

the cell membrane if it is coiled in the shape of an -helix.

Monoamine oxidase from the mitochondrial outer membrane is another typical

single transmembrane–segment protein (Figure 9.11); this enzyme is the target for

many antidepressant and neuroprotective drugs Each monomer of the dimeric

protein binds to the membrane through a C-terminal transmembrane -helix.

Residues in two loops (Pro-109 and Ile-110 in the 99–112 loop and Phe-481,

Leu-482, Leu-486, and Pro-487 in the 481–488 loop) also provide nonpolar residues that

participate in membrane binding

Approximately 10% to 30% of transmembrane proteins have a single helical

transmembrane segment In animals, many of these function as cell surface

recep-tors for extracellular signaling molecules or as recognition sites that allow the

im-mune system to recognize and distinguish cells of the host organism from invading

Leu Ser Ser Thr ThrGluGlyVal

Ala Met His ThrThr Thr Ser Ser Val Ser Ser Lys

Ser Tyr Ile Ser Ser Gln Thr Asn Asp Thr

Lys His

Arg Asp Thr Tyr Ala Ala Thr Pro Arg Ala His Glu Val

Ser Glu Ile Ser Val Arg Thr

Ile Ser Tyr GlyIle Arg Arg Leu Ile Lys Lys Ser Pro Ser Asp Val Lys Pro Leu

Leu Leu Ile Thr

Ile Leu Thr Ile Glu

Glu Pro Ser Phe His His Ala Leu Gln Val Arg Glu Gly

Thr Glu Glu Glu Pro Pro TyrVal

Gly Ala Met Val Gly Ile Val

Gly Phe Ile

Pro Ser Pro Asp Thr Asp Val Pro Leu Ser Ser Val Ile Glu Asn

Glu Pro

Thr Ser Asp Gln

COO–

Glu

H3+N—

10 20

30

40

50 60

70

100

110

120 90

130

Outside

Inside

Carbohydrate

FIGURE 9.10 Glycophorin A spans the membrane of the human erythrocyte via a single -helical

transmem-brane segment The C-terminus of the peptide faces the cytosol of the erythrocyte; the N-terminal domain is extracellular Points of attachment of carbohydrate groups are indicated by triangles.

foreign cells or viruses The proteins that represent the major transplantation antigens

H2 in mice (Figure 9.11) and human leukocyte associated (HLA) proteins in humans are

members of this class Other such proteins include the surface immunoglobulin

recep-tors on B lymphocytes and the spike proteins of many membrane viruses The function

of many of these proteins depends primarily on their extracellular domain; thus, the segment facing the intracellular surface is often a shorter one

Proteins with Multiple Transmembrane Segments Most integral transmembrane

proteins cross the lipid bilayer more than once These multi-spanning membrane

proteins typically have 2 to 12 transmembrane segments, and they carry out a vari-ety of cellular functions (Figure 9.12) A well-characterized example of such a

pro-tein is bacteriorhodopsin (Figure 9.13), which clusters in purple patches in the

membrane of the archaeon Halobacterium halobium The name Halobacterium refers

to the fact that this prokaryote thrives in solutions having high concentrations of

(b) (a)

Outside

Inside

Outside

Inside

FIGURE 9.11 (a) Major histocompatibility antigen

HLA-A2 (pdb id  1JF1) and (b) monoamine oxidase

(pdb id  1GOS) are membrane-associated proteins

with a single transmembrane helical segment.

90 100

80 70 60 50 40 30 20 10 0

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Number of transmembrane helices

FIGURE 9.12 Most membrane proteins possess 2 to 12 transmembrane segments Those involved in transport functions have between 6 and 12 transmembrane segments (Adapted from von Heijne, G., 2006 Membrane-protein

sodium chloride, such as the salt ponds of San Francisco Bay Halobacterium carries

out a light- driven proton transport by means of bacteriorhodopsin, named in

ref-erence to its spectral similarities to rhodopsin in the rod outer segments of the

mammalian retina The amino acid sequence of bacteriorhodopsin contains seven

different segments, each about 20 nonpolar residues in length—just the right size

for an -helix that could span a bilayer membrane (Twenty residues times 1.5 Å per

residue equals 30 Å.)

Bacteriorhodopsin clusters in symmetric, repeating arrays in the purple

mem-brane patches of Halobacterium, and it was this orderly, repeating arrangement of

proteins in the membrane that enabled Nigel Unwin and Richard Henderson in

1975 to determine the bacteriorhodopsin structure The polypeptide chain crosses

the membrane seven times, in seven -helical segments, with very little of the

pro-tein exposed to the aqueous milieu The bacteriorhodopsin structure became a

model of globular membrane protein structure Many other integral membrane

proteins contain numerous hydrophobic sequences that, like those of

bacterio-rhodopsin, form -helical transmembrane segments

Membrane Protein Topology Can Be Revealed by Hydropathy Plots The

topol-ogyof a membrane protein is a specification of the number of transmembrane

seg-ments and their orientation across the membrane The topology of a

transmem-brane helical protein can be revealed by a hydropathy plot based on its amino acid

sequence If a measure of hydrophobicity is assigned to each amino acid (Table

9.1), then the overall hydrophobicity of a segment of a polypeptide chain can be

es-timated The hydropathy index for any segment is an average of the

hydrophobic-ity values for its residues

The hydropathy index can be calculated at any residue in a sequence by

averag-ing the hydrophobicity values for a segment surroundaverag-ing that residue Typically,

segment sizes for such calculations can be 7 to 21 residues With a 7-residue

seg-ment size, the calculation of hydropathy index at residue 10 would average the

val-ues for residval-ues 7 through 13 The calculation for a 21-residue segment around

residue 100 would include residues 90 to 110 A polypeptide segment

approxi-mately 20 residues long with a high hydropathy index is likely to be an -helical

transmembrane segment A hydropathy plot for glycophorin (Figure 9.14a) reveals

a single region of high hydropathy index between residues 73 and 93, the location

of the -helical segment in this transmembrane protein (Figure 9.10) A

hydropa-thy plot for rhodopsin (Figure 9.14b) reveals the locations of its seven -helical

transmembrane segments Rhodopsin, the light-absorbing pigment protein of the

eye, is a member of the G-protein–coupled receptor (GPCR) family of membrane

proteins (see Chapter 32)

Proline Residues Can Bend a Transmembrane ␣-Helix Transmembrane -helices

often contain distortions and “kinks”—more so than for water-soluble proteins As

more integral membrane protein structures have been determined, it has become

clear that most transmembrane -helices contain significant distortions from ideal

helix geometry Helix distortions may have evolved in membrane proteins because

(1) helices, even distorted ones, are highly stable in the membrane environment,

and (2) helix distortions may be one way to create structural diversity from the

sim-ple helix building blocks of most membrane proteins

About 60% of known membrane helix distortions are kinks at proline residues

(Figure 9.13) Proline distorts the ideal -helical geometry because of steric conflict

with the preceding residue and because of the loss of a backbone H bond

Proline-induced kinks create weak points in the helix, which may facilitate movements

re-quired for transmembrane transport channels

Amino Acids Have Preferred Locations in Transmembrane Helices

Transmem-brane protein sequences and structures are adapted to the transition from water on

one side of the membrane, to the hydrocarbon core of the membrane, and then to

water on the other side of the membrane The amino acids that make up

trans-FIGURE 9.13 Bacteriorhodopsin is composed of seven transmembrane-helical segments connected by short

loops (pdb id  1M0M) Nearly all of this protein is embedded in the membrane Only the short loops con-necting helices are exposed to solvent A retinal chro-mophore (a light-absorbing molecule, shown in blue) lies approximately parallel to the membrane and between the helical segments A proline residue (red) induces a kink in one of the helical segments (green).

Side Chain Hydropathy Index

*From Kyte, J., and Doolittle, R., 1982 A simple method for

dis-playing the hydropathic character of a protein Journal of

Molecular Biology 157:105–132.

Hydropathy Scale for Amino Acid Side Chains in Proteins*

Go to CengageNOW at www.cengage.com/login and click BiochemistryInteractive to explore the structure

of the bacteriorhodopsin.

TABLE 9.1

membrane segments reflect these transitions Hydrophobic amino acids (Ala, Val, Leu, Ile, and Phe) are found most often in the hydrocarbon interior, where charged and polar amino acid almost never reside (Figure 9.15b) Charged residues (Figure 9.15a) occur commonly at the lipid-water interface, but positively charged residues are found more often on the cytoplasmic face of transmembrane proteins Gunnar von Heijne has termed this the “positive inside rule.” Tryptophan, histidine, and tyrosine are special cases (Figure 9.15c) These residues have a mixed character, with nonpolar aromatic rings that also contain polar parts (the ring NOH of Trp and the substituent OOH of Tyr) As such, Trp and Tyr are found commonly at the lipid–water interface of transmembrane proteins

The amino acids Lys and Arg frequently behave in novel ways at the lipid–water interface Both of these residues possess long aliphatic side chains with positively charged groups at the end In many membrane proteins, the aliphatic chain of Lys

or Arg is associated with the hydrophobic portion of the bilayer, with the positively

0

–2

4

2

120 100 80

Hydrophobic transmembrane segment

60 40 20

Residue number

0

–2

2 4

300 250

200 150

1 2

7

100 50

Residue number

FIGURE 9.14 Hydropathy plots for (a) glycophorin and (b) rhodopsin Hydropathy index is plotted versus

residue number At each position in the polypeptide chain, the average of hydropathy indices for a certain

number of adjacent residues (eight, in this case) is calculated and plotted on the y-index, and the number of the residue in the middle of this “window” is shown on the x-axis.

(b)

45 Distance from membrane center (Å)

(c)

45 Distance from membrane center (Å)

(a)

45 Distance from membrane center (Å)

FIGURE 9.15 Amino acids have distinct preferences for different parts of the membrane The graphs show

rela-tive stabilization energies as a function of location in the membrane for (a) Arg, Asp, Glu, Lys, Asn, Gln, and Pro;

(b) Ala, Gly, Ile, Leu, Met, Phe, and Val; and (c) His, Tyr, and Trp Polar and charged residues are less stable in the

membrane interior, whereas nonpolar residues tend to be more stable in the membrane interior The stability profiles for His, Tyr, and Trp are more complex.(Adapted from von Heijne, G., 2006 Membrane-protein topology Nature