Advanced Chemical Biology
Lipids
Hwan-Ching Tai & Chung-Han Chu
2019.10.28 (Mon.)
Lipids
DNA
mRNA
transcription
Protein
translation
replication
•
Sugars and lipids are outside of the confines of the central dogma. Textbooks
spend less time on these molecules; however, they are just as important as
nucleic acids and proteins.
•
Definition – naturally occurring hydrophobic small molecules
•
Lipid is a general term, which includes all of the following molecules:
–
–
–
–
–
Waxes
Mono- / di- / triglycerides
Phospholipids
Sphingolipids
Sterols … etc.
Waxes
•
•
A diverse class of hydrophobic compounds
Natural waxes are often a mixture produced by plants or animals
•
One of the main components in typical natural wax is ester form between
a fatty acid and a fatty alcohol.
mp (oC)
63
86
46
http://www.uwplatt.edu/~sundin/363-7/image/l637-35a.gif
Fatty acid and glycerol
+
Glycerol
1,2,3-protanetriol
R
R
R
R
Fatty acid
Triglyceride
•
Glyceride are esters formed between glycerol
and fatty acids
•
Glycerol can be esterified with one, two, or
three fatty acids that are often different in
natural glycerides.
Triglycerides
•
•
Plant oil (liquid at room temp.)
Animal fat (solid at room temp.)
Phospholipids
•
•
Phospholipids have two hydrophobic tails and a hydrophilic head group.
The hydrophobic tails are often different.
•
There are mainly two types of phospholipid: glyceride and sphingolipids.
Phosphoglycerides
•
•
PA = phosphatidic acid
PE = phosphatidylethanolamine
•
•
PC = phosphatidylcholine
PI = phosphatidylinositol
•
PS = phosphatidylserine
choline
inositol
Sphingolipid
Membrane lipids classification
Category
Glycerolipid
Sphingolipid
Core
Glycerol (3 carbon with 3 hydroxyl
groups)
Sphingosine (amino alcohol with a
hydrophobic tail)
Hydrophobic
fatty acid tails
2 fatty acids (ester bond)
1 fatty acid (amide bond)
additional
Hydrophilic
head group
phosphate +
alcohol
carbohydrate
phosphate+
alcohol
carbohydrate
Nomenclature
phosphoglyceride
glycoglyceride
sphingomyelin
glycosphingolipid
Common
Subtypes
1.
2.
3.
4.
choline (PC)
ethanolamine (PE)
inositol (PI)
serine (PS)
1.
1.
2.
glucose
galactose
1.
2.
choline
ethanolamine
2.
monosaccharide
(cerebroside)
polysaccharide
with sialic acid
(ganglioside)
More on fatty acids
The most abundant hydrophobic
molecules in nature are fatty acids.
mp (oC)
mostly cis
double bond
75
70
63
What is trans fat?
•
Trans fat is made by hydrogenation of vegetable oil
•
The trans double bond does not create a kink
•
Trans fat increases the risk of cardiovascular disease
H2/ Ni cat.
250 oC
Common animal fatty acids
1. No trans fatty acid!
2. Even number of carbons?
Fatty acid biosynthesis
=
=
Acetyl-coA
•
Acetyl-coenzyme A is the “primer” in fatty acid biosynthesis
•
The Ac group is transferred from acetyl-CoA to the acyl carrier protein (ACP)
•
Malonyl-CoA is the two carbon building block
Fatty acid biosynthesis
•
The a carbon of malonate acts as the nucleophile to react with acetyl-CoA, and
the chain extends by 2 carbons after decarboxylation
•
The ketone (C=O) is reduced to a hydroxyl group (OH)
Branched fatty acids
•
Loss of water forms an unsaturated carbon double bond
•
The C=C bond is then reduced to form a saturated straight-chain fatty acid
•
There are desaturases to oxidize fully saturated fatty acids
Branched fatty acid
•
Branched fatty acid synthesis uses the
same pathway but starts with a different
“primer.”
•
Valine, leucine, and isoleucine can be
activated as an a keto-acid and then
loaded onto CoA
Cell membranes are supported by protein networks
EM image: inner membrane skeleton
16
Cellular membranes are composed of lipids and proteins
60:30:10
Protein : Lipid :
Saccharides
The membrane is a barrier; cells
need ways to communicate with
the outside world.
Membrane proteins have alternating hydrophilic/hydrophobic residues
hydrophobic residues
hydrophilic residues
Membrane proteins have alternating hydrophilic/hydrophobic residues
Kyte, J; Doolittle, RF. A simple method for displaying the hydropathic
character of a protein. J. Mol. Biol. 1982, 157, 105-132.
Channels and pumps
• Membrane is a physical barrier. Cells need to communicate with the
outside world.
• Pumps require energy to operate. Their energy can come from
– ATP hydrolysis
– Coupled to proton gradient
• Channels are passive throughways
– No energy required for transport
– Ions/molecules diffuse in accordance to the concentration gradient
– Some channels are highly selective
:: Water channel impermeable to ions
:: Potassium (K+) channel impermeable to sodium (Na+)
:: Glucose (Glc) channel impermeable to galactose (Gal)
• 67% of FDA approved drugs target membrane proteins.
The human protein atlas: https://www.proteinatlas.org/search/protein_class%3AFDA+approved+drug+targets/2
Lipid rafts are membrane microdomains
Inside
Outside
1. Non-raft membrane
2. Lipid raft
3. Lipid raft associated transmembrane protein
4. Non-raft membrane protein
5. Glycosylation modifications (proteins and lipids)
6. GPI-anchored protein
7. Cholesterol
8. Glycolipid
Lipidation can direct protein destination
Opportunities for chemists
Protein / lipid engineering
• Target protein toward a defined
location (raft) in the membrane
Table: lipid modifications on proteins
• Generate an entirely new lipid
domain (raft).
– e.g., fluorinated lipids
Buer, BC; Marsh, EN. Fluorine: A new element in
protein design. Protein Sci. 2012, 21, 453-462.
Nadolski, MJ; Linder, ME. Protein lipidation.
FEBS J. 2007, 274, 5202–5210
Sterols and steroids
C
D
A
B
Steroid backbone
Sterol
Cholesterol
• Cholesterol is mostly found in the cell membrane.
• Cholesterol enhances the packing of membrane lipids, resulting in
reduced membrane fluidity and permeability (bad).
• Cholesterol is the biosynthetic precursor to many steroid hormones
– Estrogen (female sex hormone)
– Testosterone (male sex hormone)
– Glucocorticoid (reduces inflammation)
The isoprene building block
• All animal cells manufacture cholesterol
• The isoprenyl pyrophosphate (IPP) building block comes from
melavonic acid.
• Multiple isoprene units are joined linearly
https://en.wikipedia.org/wiki/Cholesterol#Biosynthesis
Steroid biosynthesis – cascade reactions
Yoder, RA; Johnston, JN. A case study in biomimetic total
synthesis. Chem. Rev. 2005, 105, 4730-4756.
Cholesterol is the precursor for steroid hormones
Organelles are often enclosed by membrane
• A membrane enclosed
compartment creates a “reaction
chamber” that confers
selectivity and increases the
reaction rate.
• Physical barrier >> selectivity
• Higher effective concentration
>> increased reaction rate
• Quiz 1: Estimate the thickness
of the cell membrane.
• Quiz 2: Estimate the approx.
concentration of a single
molecule in a mammalian cell,
an E. coli, and an organelle.
http://media.wiley.com/Lux/36/8536.nfg001.jpg
Quiz 1 and 2 notes
Endoplasmic reticulum (ER)
1
1
2
2
2
[Stryer, Biochemistry] Figure 12.36 Nuclear envelope. The nuclear
envelope is a double membrane connected to another membrane
system of eukaryotes, the endoplasmic reticulum (ER).
The mitochondrion
• Mitochondria have two membranes (inner/outer)
and a intermembrane space
• It may have been a symbiotic prokaryotic cell
living inside the eukaryote.
Stryer, Bicochemistry, Fig. 18.2
Opportunities for chemists 1
Erdmann et al.
Author Manuscript
• Lipids as a handle for
labeling / modification
• Unique compartment
• Origin of life studies
Labeling the Golgi apparatus via lipids
for super-resolution (SR) imaging
• SR imaging needs very robust
fluorophores (or a lot of them)
Author Manuscript
• There are a lot more lipid molecules
than proteins for saccharides
Figure 1.
Autho
Two-step procedure for high-density labeling of the Golgi in living cells. Cells are tre
Erdmann, RS et al. Super-resolution imaging of the Golgi in live cells with a biofirst
with Cer-TCO,
a trans-cyclooctene-containing
lipid,
then reacted w
orthogonal
ceramide
probe. Angew. Chem. Int.ceramide
Ed. 2014,
53,and
10242-10246.
Opportunities for chemists 2
• Lipids as a handle for
labeling / modification
• Unique compartment
• Origin of life studies
Direct micelle / vesicle (liposome)
formation using different lipids
• Conical lipids
Micelle
Liposome / vesicle
• Cylindrical lipids
http://bioweb.wku.edu/courses/biol22000/2Bonds/images/F02-20.JPG
Opportunities for chemists 3
• Lipids as a handle for
labeling / modification
Vesicle composition / formation:
• Unique compartment
• Homogeneous vs. heterogeneous;
Synthetic vs. biological
• Origin of life studies
• Physical manipulation
Elani, Y et al. Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules.
Sci. Rep. 2018, 8, 4564.
Chen, IA; Walde, P. From self-assembled vesicles to protocells. Cold Spring Harb. Perspect. Biol. 2010, 2, a002170
Opportunities for chemists 4
• Lipids as a handle for
labeling / modification
• Unique compartment
• Origin of life studies
A diverse array of vesicles
• Small unilamellar vesicle (SUV)
• Large unilamellar vesicle (LUV)
• Multilamellar vesicle (MLV)
• Multivesicular vesicle (MVV)
http://bioweb.wku.edu/courses/biol22000/2Bonds/images/F02-20.JPG
systems which are able to couple molecular-based template selfreplication to cellular objects evolved, overcoming parasitism and
Replication
problems of dilution, see Fig. 11. To achieve this we propose a
cycle
strategy that combines the property of molecular replication with
that of the formation of a protective bounce of growth and fission,
and ultimately objects that could undergo cycles of Darwinian
T
A B
T
evolution2, 3. Such ‘model protocells’ would allow the idea that life
on earth emerged from a minimal self-organised cell-like entity—
c
b
Lipidstoas
handle
for 2.
a• protocell,
be a
tested
experimentally
4
Template self-replication relies on binding of the precursors to
labeling / modification
the template by molecular recognition, usually combining a
H2O
hydrogen
bonding
motif with size, shape and/or charge com•plementarity.
Unique
compartment
The process may be straightforward and specific, as
Amphiphilic
in
the
minimal
template
self-replicator
model
or
more
complex,
replicator
• Origin of life studies
as is the case for the replication of DNA. There are numerous
examples of artificial replicators that use molecular recognition to
Reverse micelle stabilization
Droplet fission
ICATIONS
| DOI:
10.1038/s41467-017-00177-4
Various
forms
of vesicle
“protocells”
catalyse
their
own formation
by an
autocatalytic cycle, but
coupling these to other functions that would help the process of Fig. 1 Formation of an amphiphile from a template self-replication
4–6. Howeverofwethe
• Autocatalytic
replication
lipid that reaction20. a Reactants A and B contain complementary recognition sites
hypothesised
evolution
is a big challenge
since a reaction that produces an amphiphile can result in the and react together via a bimolecular reaction to form template T. T
self-assembly of supramolecular structures such as micelles or selectively binds A and B by the corresponding recognition sites, giving a
vesicles7, the useOof a template self-replication reaction to produce ternary complex [ABT] where the reactive groups of A and B are perfectly
O
an amphiphile would linkN self-replication
to the formation ofH2Oplaced to react together, resulting
O
in a rapid pseudounimolecular reaction.
N
O
N
O
H
such structures.
Often
the
starting
materials
for
these
amphiphile
The template dimer [TT] can then
dissociate to give Otwo molecules
of T,
H
N
N
H
H
N
H b Reactants A and B
formation are Hhydrophobic
(e.g. ethyl Ncaprylate
in its hydrolysis completing the autocatalyticN cycle.
H are Nonly weakly
O
H
H
O
H
the reaction benefits from theirH Oamphiphilic, but by bringing together
to caprylic acidO8) and therefore
N
a hydrophobic and Na hydrophilic
2
O
N
O
solubilisation in micelles, resulting
in self-reproduction of the group, amphiphilic T is formed. This is demonstrated
by Oits ability to
O
micelles (autopoiesis). Autopoiesis is thought to be a key step in stabilise reverse micelles of water in chloroform. c Stabilisation of a droplet
[3•3]
the origin of life due to the necessity
of a compartment to prevent chloroform/water interface by amphiphilic T allows
droplets to undergo
[1•2•3]
Autocatalytic
molecular information dispersing into the bulk solution, which in fission, as demonstrated by placing microlitre volume droplets from a
cycle9, 10 sample of a reaction mixture periodically using a liquid-handling robot
turn allows individuals to evolve. A minimal protocell
therefore requires compartmentalisation, self-replication (to transNH
ferH information
from one generation to the next) and metabolism
O
OH
C H
N
The unseeded reaction gave a sigmoidal concentration vs. time
(toN utilise material and energy from the environment for growth).
H2O
3
O
with an induction period characteristic
of autocatalytic
Herein
1 we present our studies on the effects of compartmen- profile
N
reactions,
as
initially
the
concentration
of
template
was low, and
talisation on a template-based self-replicator where the template
H
N
N
the3 majority of the template was formed
by the bimolecular
Template
is amphiphilic, and we observe the effects of a self-replication
H2O an oil-in-water droplet in a search Ofor
reaction of 1 and 2. As [3] increased,dissociation
the rate also increased to a
reactionO physically on
O
OH emergent system properties. The amphiphilicity of the template is
maximum as the reaction increasingly took place via a [1·2·3]
2
Bimolecular
investigated by
dynamic light scattering (DLS), and periodic ternary complex. With the seeded reaction, enough 3 was present
sampling of self-replication
reaction reaction mixtures yields an increase at the start of the reaction to ensure the maximum rate was
reached
at the start
the reaction,
with
sigmoidal
behaviour. model
in fission of the droplets after placement into an aqueous phase
as JW;
Taylor,
Eghtesadi,
SA;ofPoints,
LJ; Liu,
T; no
Cronin,
L. Autonomous
Both
curves
were
fitted
to
an
adaptation
of
the
minimal
replicator
the reaction progresses.
protocell division driven by molecular replication. Nat. Commun.
2017, 8, 237.
Opportunities for chemists 5
2
12 25
11
Opportunities for chemists 6
• Lipids as a handle for
labeling / modification
• Unique compartment
• Origin of life studies
Various forms of vesicle “protocells”
• Replication of nucleic acids inside the
protocell
– Mg2+ is required for RNA replication
but it also promotes RNA and protocell
degradation.
– Discovered citrate as a chelator that
preserves Mg2+ function but
suppresses degradation.
• Growth and division of vesicle protocells
(movie link)
Adamala, K; Szostak, JW. Nonenzymatic template-directed RNA
synthesis inside model protocells. Science 2013, 342, 1098-1100.
Zhu, TF; Adamala, K; Zhang, N; Szostak, JW. Photochemically
driven redox chemistry induces protocell membrane pearling and
division. Proc. Acad. Natl. Sci. 2012, 109, 9828-9832.
A seminar you should not miss
Daniel Kahne
KT Wang Lectureship
12/18 (Wed.)
Molecular machines that
make membranes
Assigned reading materials
REVIEWS
Membrane lipids: where they are
and how they behave
Gerrit van Meer*, Dennis R. Voelker‡ and Gerald W. Feigenson§
Abstract | Throughout the biological world, a 30 Å hydrophobic film typically delimits the
environments that serve as the margin between life and death for individual cells.
Biochemical and biophysical findings have provided a detailed model of the composition and
structure of membranes, which includes levels of dynamic organization both across the lipid
bilayer (lipid asymmetry) and in the lateral dimension (lipid domains) of membranes. How do
cells apply anabolic and catabolic enzymes, translocases and transporters, plus the intrinsic
physical phase behaviour of lipids and their interactions with membrane proteins, to create
the unique compositions and multiple functionalities of their individual membranes?
Triacylglycerol
A family of storage lipids
consisting of glycerol esterified
to three fatty acids, forming the
hydrophobic core of lipid
droplets and blood
lipoproteins together with
steryl esters.
From the ongoing cataloguing of lipid structures (lipidomics), it is clear that eukaryotic cells invest substantial
resources in generating thousands of different lipids1. Why
do cells use ~5% of their genes to synthesize all of these
lipids? The fundamental biological maxim that ‘structure
subserves function’ implies that there must be evolutionary advantages that are dependent on a complex lipid
fission and fusion, characteristics that are essential for
cell division, biological reproduction and intracellular
membrane trafficking. Lipids also allow particular proteins in membranes to aggregate, and others to disperse.
Finally, lipids can act as first and second messengers in
signal transduction and molecular recognition processes.
The degradation of amphipathic lipids allows for bipartite