Table of Contents
Fundamentals of Biochemistry
Biochem 380 - Fall 2006
Lecture 027
Outline
- Announcements
- Questions from previous lecture
- Section 9.8: Biological Membranes
- Section 9.9: Membranes Are Dynamic
- Section 9.10: Membrane Proteins
Announcements
- Lecture 26 notes are online
Questions
- Any questions on the material from the previous lecture?
Section 9.8: Biological Membranes
- In Chapter 2, we first discussed the amphipathic nature of detergent molecules and their ability to
self-assemble into monolayers and micelles
- Biological lipid molecules such as glycerophospholipids and glycosphingolipids can also
form monolayers under certain conditions, but in cells they form lipid bilayers:
- These are flexible and self-sealing membranes about 5 to 6 nm thick that consist of two sheets
(also called leaflets). They are maintained by the noncovalent interactions between lipid molecules
Composition of Cell Membranes
- Lipids form the main framework for cell membranes. Lipids may form anywhere from 25% to 50%
of the total content in a typical membrane. The remainder consists of protein (50% to 75%), with less
than 10% as carbohydrates
- The precise composition varies, depending upon species and cell type. For example, the myelin sheaths
in nerve cells are mostly lipid, while the inner mitochondrial membrane is mostly protein
Electron Micrograph of the Lipid Bilayer
- The composite nature of the cell membrane can be seen in the following electron micrograph,
which is produced by a freeze-fracture technique. The rough surface of the inner leaflet
is a result of proteins embedded in the lipid layer:
Models of Cell Membranes
- Our understanding of the structural and dynamical properties of cell membranes is still being developed
- The current understanding of cell membranes is the result of a succession of models that have been developed
over the years to explain the properties and features that have been observed
- It was known for some time that cell membranes contained a lipid bilayer, in association with proteins
- However, earlier models of membrane structure proposed that these components were organized as a
lipo-protein sandwich, in which the proteins formed an outer layer around the interal lipid bilayer
The Fluid Mosaic Model
- These earlier models were subsequently modified by Singer and Nicolson in 1972, with their
proposal of the fluid mosaic model to explain the organization of biological membranes
- The fluid mosaic model proposes that integral membrane proteins are 'dissolved' within the lipid bilayer, which
acts as both a two-dimensional solvent and a permeability barrier:
- Although the fluid mosaic model accounts for many of the features observed in membranes,
additional factors are needed to explain their behavior
- These factors include interactions with the cytoskeleton and the dynamic formation of rigid structures
such as clathrin cages, which are involved in transport processes such as endocytosis
Section 9.9: Membranes Are Dynamic
- The fluid nature of cell membranes allows for constant synthesis and motion of membrane components, in a variety
of ways. We'll consider some of these dynamic aspects of membranes including:
- Membrane diffusion: lateral and transverse
- Lipid diffusion rates
- Melting temperatures of fatty acids
- Adjustment of membrane composition to changes in temperature
Membrane Diffusion
- The lipid bilayer in cell membranes can be thought of as a two dimensional fluid
- The lipid molecules that form the bilayer are held together by non-covalent interactions such as
van der Waals forces, hydrogen bonding and hydrophobic effects
- As with other liquids, these associations are only temporary, and an individual lipid molecule is
free to move within the plane of the membrane
- This property, lateral diffusion, is also observed in proteins embedded in the membrane, although
the rates of protein diffusion can vary, with some being virtually immobile
- The fluid properties of membranes are affected by temperature, and cells will change the ratios of
lipids types in order to adjust to temperature fluctuations
FRAP
- The lateral movement of components in cell membranes can be visualized with an experimental
technique called fluoresence recovery after photobleaching (FRAP)
- The technique works by attaching fluorescent molecules to lipids within the membrane and
then observing the fluorescent intensity in a microscope:
- The intensity in a particular region can be temporarily reduced by bleaching, which results
in destruction of the fluorescent molecules in that region by a laser pulse
- After bleaching, the intensity will return to the previous level as a result of diffusion of other
labelled molecules back into the region
Membrane Diffusion Rates
- Using techniques such as FRAP, the diffusion rate of membrane molecules can be determined quantitatively
- The average distance s traversed in time t can be calculated from the diffusion coefficient
D, according to the following equation:
s = (4Dt)0.5
- A typical diffusion coefficient value for lipid molecules is D = 1 μm2/sec
- Consequently, for t = 1 sec, a lipid molecule will diffuse an average distance of
s = [(4)(1 μm2/sec)(1 sec)]0.5 = 2 μm
Membrane Diffusion Rates
- For bacterial cells on the order of 1 μm in length, this means that an individual lipid molecule can diffuse
from one side of the cell to the other in about 1 second
- The corresponding viscosity of the lipid bilayer is about 100 times that of water, similar to olive oil
- The diffusion rates of proteins in the membrane can vary greatly
- For example, the photoreceptor protein rhodopsin is very mobile, with a diffusion coefficient of
0.4 μm2/s
- In contrast, the peripheral glycoprotein fibronectin is virtually immobile, with D less than
10-4 μm2/s
- The mobility of membrane proteins is a function of their mass, as well as other factors such as interactions
with intracellular or extracellular molecules, which can anchor or restrict movements in the membrane layer
Lateral and Transverse Diffusion
- Although the lateral diffusion of many components in the membrane can be quite rapid, the transverse
diffusion or flip-flop of lipid molecules from one layer to the other occurs much more slowly:
- Measurements of phosphatidyl choline vesicles have shown that a phospholipid molecule will flip-flop
about once every several hours
- This means that it takes phospholipids about 109 (a billion) times as long to
flip-flop transversely as it does to diffuse 50 A in the lateral direction
- The energy barrier for transverse diffusion of proteins is larger than phospholipids and spontaneous
flip-flops of proteins have not yet been observed
Melting Temperature
- Because lipid membranes act like two-dimensional liquids, they can also undergo phase-transitions
at distinct temperatures:
- The transition from a rigid to a fluid state occurs fairly abruptly as the temperature is raised
- The melting temperature, Tm, at which such transitions occur is influenced by the length
and degree of saturation of fatty acids in the membrane
Fatty Acid Melting Temperatures
- The quantitative effect of chain length and saturation can be seen from a comparison of melting temperatures
for different fatty acids in the following table:
Dynamic Adjustment of Membrane Composition
- Because properties such as diffusion rates in cell membranes are important for many processes such as
transport and signaling, organisms will change the composition of membranes to adjust to temperature fluctuations
- Just as people respond to temperature fluctuations by changing their clothing, organisms can
vary the number of double bonds and lengths of fatty acyl chains used in the lipid bilayer
- For example, E. coli will decrease the ratio of saturated to unsaturated fatty acyl chains from
1.6 to 1.0 as the temperature changes from 42°C to 27°C
- This prevents the membrane from becoming too rigid at the lower temperature
Section 9.10: Membrane Proteins
- The lipid components provide the core barrier properties of a cell membrane, but it is the protein
components that enable the many kinds of selective permeability that are found in lipid bilayers
- The distribution of membrane proteins will vary with cell type, as can be seen by comparison of
SDS-PAGE separations:
- The three bands reflect the different membrane proteins found in the plasma membrane of erythrocytes (A),
photoreceptor membranes in retinal cells (B), and the sarcoplasmic reticulum of muscle cells (C)
- The various types of membrane proteins include pumps, channels, receptors and enzymes
Integral and Peripheral Membrane Proteins
- A distinct difference is observed in the strength of association of a protein with the cell membrane
- Some proteins can be dissociated fairly easily by strong ionic solutions
- Others can only be removed with much harsher treatments such as detergents or organic solvents that
will disrupt the lipid bilayer
- These differences are used to categorize membrane proteins into two kinds: integral membrane proteins
and peripheral membrane proteins
Integral and Peripheral Membrane Proteins
- A schematic representation of proteins in these two categories are shown below:
- The integral membrane proteins a, b, and c, shown in yellow, all have extensive hydrophobic interactions
with the lipid interior, as a result of membrane-spanning segments contained within these proteins
- The peripheral membrane proteins e and d, shown in blue, maintain their association through
electrostatic and hydrogen bond interactions with other molecules at the periphery of the membranes
Membrane Protein Structure
- Determining the 3D atomic structure of membrane proteins is considerably more difficult for soluble
proteins, mostly because of difficulties in obtaining good crystal structures suitable for x-ray diffraction
- As a result, our current knowledge of membrane proteins is much less extensive compared to aqueous proteins
- Recent successes have provided some details of the structures and associations of membrane proteins
- We'll look at two examples:
- Bacteriorhodopsin, a membrane-spanning protein with a seven α-helix motif
- Porin, a channel protein formed by a β-barrel
Bacteriorhodopsin is an Alpha Helical Membrane Protein
- Bacteriorhodopsin is an archael protein that transduces light energy into proton transport across
a plasma membrane
- It is almost entirely α-helical, with 7 α helices that span membranes from one side to the other:
- This 7 helical pattern is a common structural motif seen in other kinds of proteins
- The particular pattern of amino acids that are required to form alpha helices that span hydrophobic
regions allows for the prediction of such structural motifs in hydropathy plots
Hydropathy Plots
- These quantitative values can then be used to create a hydropathy plot, in which each point
on the x axis is a summation of free energy changes for a subsequence of amino acid residues:
- The first plot indicates the existence of a membrane-spanning segment because of the crossing of an energetic threshold
- The second plot is for a protein which does not have this property
Prediction of Transmembrane Helices
- The structural constraints of membrane-spanning proteins with α-helical segments enables their
prediction from analysis of the primary structure (amino acid sequence)
- The method is based upon the constraint that a subsequence of approximately 20 amino acid residues
must contain hydrophobic side chains that span the lipid interior:
- This constraint is used in conjunction with a quantitative table of the differences in hydrophobicity
of the 20 amino acid side chains that can be determined using thermodynamic methods
Porin is a Beta Barrel Membrane Protein
- A bacterial porin protein from Rhodopseudomonas blastica is a membrane protein with a completely
different structural organization
- It composed entirely of β strands that wrap around to form a barrel or hollow cylinder:
- The amino acid sequence of the residues in the beta strands also exhibit a particular pattern that is determined by the need to
have hydrophobic residues on the outer face of the barrel and hydrophilic residues on the interior face
Liposomes
- The spontaneously assembly of phospholipid structures is not limited to formation of bilayer sheets
- Under the appropriate conditions, phospholipids can form bilayer structures that will enfold into
spherical structures with internal aqueous compartments:
- Such structures are called lipid vesicles or liposomes
- They can be formed in a number of ways, such as through sonication of a mixture of phospholipids and water,
or through evaporation of organic solvent from a mixed suspension
- Liposomes of characteristic sizes are produced (50 nm, 1 μm), depending on the procedure
Liposomes Can Encapsulate Other Material
- Liposomes are currently an area of great interest because of their potential use in drug delivery systems
- It is possible to prepare liposomes in a manner that results in encapsulation of other molecules within
the aqueous interior:
- For example, sonication of phospholipids in a solution of glycine, followed by subsequent removal of the free
glycine, results in lipid vesicles containing a 'payload' of trapped glycine
- Although many difficulties remain to be worked out, if artificial vesicles can be prepared as drug containers
in a manner that enables them to be delivered with efficiency and specificity to target cells, the
therapeutic potential is enormous
Liposomes and Drug Delivery
- The use of liposomes as vehicles for drug delivery has been a subject of research for the last 25 years.
Because they have both aqueous and lipid components, they have the potential to act as carriers for both
lipophilic and hydrophilic drugs
- Lipsomes might be able to be introduced either topically through the skin, or by injection into the bloodstream.
Since conventional liposomes are often rapidly cleared from the blood through phagocytosis, additional
modifications have been made to enhance their half-life, such as by pegylation (addition of PEG, Polyethylene glycol)
- Another potential benefit of liposomes is the reduction of toxicity compared to conventional drug
delivery. For example, liposomal doxyrubicin, used for treatment of ovarian
cancer and Kaposi's sarcoma, has reduced toxicity compared to non-liposomal forms of the drug
Questions
- Questions about the material covered today?
References
Liposomes
- Nano- or Submicron-Sized Liposomes as Carriers
for Drug Delivery,
Jia-You Fang, PhD,
Chang Gung Med J Vol. 29 No. 4
July-August 2006
- Emerging use of nanoparticles in diagnosis and treatment of breast cancer,
Maksym V Yezhelyev, Xiaohu Gao, Yun Xing, Ahmad Al-Hajj, Shuming Nie, Ruth M O’Regan,
http://oncology.thelancet.com Vol 7 August 2006
Next Lecture: Sections 9.11 - 9.12
- Read Sections 9.11 - 9.12
