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“There are many more things in
heaven and earth, Horatio, than
are dreamt of in your philosophy.”
Hamlet, Act 1, Scene 5
Biochemistry
Medicine
The Great
Unknown
E. coli
“to scale” artwork by David Goodsell
Red Blood Cell and Blood Serum
Biomolecular Scale
1 Angstrom = 0.1 nm
atoms and
bonds:
1-3 Angstrom
typical single
domain globular
protein:
35-50 Angstroms
in diameter
Classes of Biomolecules
Fats, Oils, Lipids
Co-Factors (Metal ions, vitamin derivatives)
Carbohydrates
Metabolites
Nucleic Acids
Proteins
Minimal Ingredients for Life on Earth?
•liquid water
•physical scaffold
•glue
•organization
•semi-permeable barriers
•source of energy
•capacity to harvest, store, and transduce energy
•information storage
•information copying, and retrieval
•capacity for sensing environment
•logic for information use, integration, and response
•capacity for regulation (switches and throttles)
•catalysts
•systems for quality control: error management and
exploitation
•capacity for reproduction
Minimal Ingredients for Life on Earth?
Proteins
play major,
usually
dominant,
roles in…
•liquid water
•physical scaffold
•glue
•organization
•semi-permeable barriers
•source of energy
•capacity to harvest, store, and transduce energy
•information storage
•information copying, and retrieval
•capacity for sensing environment
•logic for information use, integration, and response
•capacity for regulation (switches and throttles)
•catalysts
•systems for quality control: error management and
exploitation
•capacity for reproduction
Proteins are linear polymers
of amino acids
• 20 different amino acids: many combinations
• Proteins are made in the RIBOSOME
amino acids
ribosome
protein
(short proteins are called
petides or polypeptides)
How Significant are the Roles of Proteins in Human Disease?
Consider “simple” inherited diseases…
5000 known simple disorders.
Mutations involving about 2000 different genes
have been documented.
53,000 mutations now logged
What are the consequences of these known mutations?
Heritable Genetic Lesions Logged
Heritable Mutations Linked to “Simple” Diseases
7000
Human Gene Mutation Database (Cardiff)
www.hgmd.org
6000
5000
4000
3000
2000
1000
1986
1988
1990
1992
1994
1996
Year
1998
2000
2002
Heritable Mutations Linked to “Simple” Diseases
53,000 Total Chromosomal DNA Mutations:
10%: involve gross DNA changes
10%: affect RNA splicing
80%: fall in protein open reading frames
of these, 30,000 are missense mutations that code for
a single amino acid change in a protein
(missense mutation = single DNA base change)
Mutations can cause protein:
loss of function
altered protein function
“gain of function”
Diabetes insipidus: a disease caused by a protein’s loss of function.
Notes on protein nomenclature:
Human Vasopressin V2 Receptor
•beginning of chain is “N-terminus”
•end of chain is “C-terminus”
•amino acids (“residues”) are
numbered from N-terminus to
C-terminus
HN
2
P
E
L
Q S S N
P L
T T
S A M L M
1
S
20
P R
D
R
C
G
A
F
A E
P
200
300
A
E
P
W
P
192 W
E V
N
G
L
G
R
C
D
D
R
R
D
G
E
A 112
D
R
W
Q
D
L
T
S
G
P
R
A
A
C
180
G
T
R
V
A
100
F
T
A
A
L
A
L 289
W
V
Y
L
I
K
P
K
Q
V
A
F
F
A
W
Y
R
L
L
T
V
V
F L 309 L
L
Q
W
Q
39 A E
209
P
Q
F
I
L
P
L
L
A
120
M
S
L
V
P
A
L
A
L M
M
V
171
G
L
L L
L
W
F
L
M
V
92 Q
L
A
L
L
F
C
S
S
Y
A
S
V
V
F
L
I V
A
S
Y
319
A
W
A
N
F
V
A
S
P
279 V
A
S
V
V
L
T
50 A
M
T
V
L V
L
V
D
I
A
I
V 160
I
A
N
G
V
L 131
A
L
L
P
P
W
A
L
M
R
S
A 220
81 L C
T
I
N
T
N
C
H
L
M
Y
W
L
R
Q
A
G
H
G
D
V
V
V L
S
I
L
A
I
R
269 T
F
A
A 60
K
H
F
F
V
L
R
A
S
G
H
R 230
V
S
A
A 140
A
S
I
E
330
R
G
I
A S
P A
I
R
H 150
W
V H
C
H
A
G
R
260
R
S
H
R
Y
P
E D Q
E G A
G
70
G
M
L
R
P
P
V
L A
E R
S
S
S
P
P
C
G T R
T
G P
G
T
240
R
G
360
G
R
R R
250
T
cytosol
S P L S
10
A S
H G P V
30
F
S
P
S
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
8-arginine vasopressin (the anti-diuretic hormone): binds
to the vasopressin V2 receptor, turning on its signaling state
ectoplasm
V
S
L
E
S
S
G
C
L
L 340
C
A
R
S
G
R
L
A S
R
S
350
S
P P
T
K
A
S
L
D T
S 370
S
-
COO
Diabetes Insipidus
and the Vasopressin
V2 receptor
aquaporin
Diabetes Insipidus
90% of cases caused by X-linked mutations
that lead to a loss of function in the
vasopressin V2 receptor.
Mostly affects males (1/250,000).
Symptoms start soon after birth: chronic
severe thirstiness, dilute urine, high blood
sodium, fever, constipation, etc.
Disease is manageable. Lots of water,
low sodium diet, and certain diuretic drugs.
(diuretic = promotes urine formation and loss
of sodium)
Protein in Complex Multi-Factorial Diseases
Proteins play central roles in 11/15 of the leading causes of death in the U.S.
How many human proteins are there?
How Many Protein-Encoding Genes Are There?
M. genitalium: 500
E. coli: 4000
M. tuberculosis: 4000
Yeast: 6000
C. elegans: 18,000
Drosophilia melanogaster: 13,000
Rice: 60,000
Humans: 22,000 (best current estimate)
Protein Sequence Defines Identity
The average human gene
contains 9 introns.
40-60% of all human genes
are expressed in multiple
splice variant forms.
There are many more
proteins than the ca. 22,000
genes that encode them.
From: Alberts
parent gene
RNA splicing,
then translation
post-translation
modifications
splice
variants
Nature often generates diversity based on combinatorial principles.
Proteins Can Be Classified According to Molecular Function
The vast majority of drugs target proteins.
Known drugs appear to target <150 proteins.
There are at least 600 potential targets.
Targets of marketed small-molecule drugs by biochemical class.
(G protein-coupled
receptors)
99% are proteins
This Concludes The
“Biomedical Significance of Proteins”
Section
Useful Observations About Protein Biochemistry…
•
Water water everywhere…
•
Temperature range of life is limited.
•
Think velcro.
•
Functional redundancy is a recurrent concept.
•
Just because you take it away and nothing happens doesn’t mean it’s not
important.
•
Reductionism is great, but it does have limits.
•
Pleiotropy is a recurrent concept.
•
Thermodynamics and kinetics matter, but you don’t have to be J. Willard
Gibbs.
•
Patterns and paradigms go a long way.
The Energetic Governance of Biomolecular Interactions
Total Energy = Kinetic Energy + Potential Energy
Energy Units
Quantities of Matter Used in PE
Joules
moles (biochemicals)
Calories
coulombs (electricity)
1 Calorie = 4.184 Joules
partial pressure (gas)
Chemical Potential Energy Units: kJ/mole or kCal/mole
Electrical Potential Energy Units: volts: (Joules/Coulomb)
Faraday’s Constant: 96,500 coulombs/mole
Kinetic energy in biology = random thermal energy =“kT energy”
Boltzmann distribution.
Brownian motion and the random walk.
From:
Krell Institute
Aquaporin
Membrane
Bilayer
Shape isn’t everything.
Experimental Electron Density Maps for the 20 Amino Acid Side Chains
What does it mean when something is at equilibrium?
R+L
RL
At equilibrium:
There is no net flux between states.
Free and bound receptor and ligand populations are constant.
forward rate = reverse rate
kforward.[R]..[L] = kreverse . [RL]
Indeed:
Keq = kforward/kreverse
The Equilibrium Constant
kforward.[R].[L] = kreverse . [RL]
[RL]
kforward/kreverse=
= Keq
[R] . [L]
R +L
RL
Keq
Position of an equilibrium is described by its equilibrium constant,
Positions determined by the free energy difference.
reactants
Energy
DGo
products
By definition:
DGo = -RTlnKeq
DGo is known as the “standard Gibbs free energy”.
Negative DGo: products favored.
Note the logarithmic relationship between energy and Keq.
Note also that being at equilibrium does not mean static.
Coupled Equilibria
R+L
RL + L
RLL
R+L
RL
Keq,1
RL + L
RLL
Keq,2
Overall equilibrium constant:
R + 2L
RLL
Keq,overall
Keq,1 x
Keq,2
=
Keq
:
DGo1 + DGo2 = DGooverall
Important Classes of Equilibria in Biological Systems
Ligand binding to a “receptor”
carrier-ligand
enzyme-substrate
antibody-antigen
protein-nucleic acid
protein-protein
Membrane partitioning (ligand in solution vs. ligand in membrane)
Keq = [Membrane-Bound Ligand]/[Ligand in Solution]
Biochemical Reactions (reactants vs. products)
[C] . [D]
For:
A+B
C+D
Keq =
[A] . [B]
Protein Folding (unfolded form versus folded form)
Keq = [Folded]/[Unfolded]
Acid/Base Buffering (the “ligand” is the proton)
Ectoplasm
N
Equilibria Involved in Signaling
By G Protein-Coupled Receptors
(such as the Vasopressin V2 Receptor)
Cytoplasm
C
hormone
(i.e., vasopressin)
K basal << 1
“off” signaling state
K activation >>> 1
“on” signaling state
“on” signaling state
In the absence of hormone, only the first equilibrium is operative, and so the receptor is mostly in its off signaling
state. When the hormone becomes abundant, the overall equilbrium shifts towards the “on” signaling state
because Kactivation is >> K basal. In other words, under normal physiological conditions, the receptor significantly
populates the signaling-active state only when the hormone is present.
In “loss of function” disorders such as diabetes insipidus, some of the causitive mutations can lower the free energy of the
“off” state to the exent that it becomes the predominate state of the receptor, even under conditions where the hormone
would normally drive the equilibrium completely towards the “on” state. In other words, the basal equilibrium constant is
reduced to be even smaller than usual.
Alternately, mutations can induce “loss of function” by mutating the hormone binding site so that the receptor does not
significantly bind the hormone at normal activating concentrations. In other words, some mutations lower the activation
equilibrium constant dramatically, making the receptor unresponsive to hormone at normally-stimulating concentrations.
Equilibria Involved in Signaling By G Protein-Coupled Receptors
How mutations can can cause constitutive “gain of function”.
hormone
(i.e., vasopressin)
K basal<< 1
“off” signaling state
K activation >>> 1
“on” signaling state
“on” signaling state
Above: Normal Conditions for Signaling
For the vasopressin V2 receptor (and a number of other related receptors), there is another class of mutations that can
change the
difference in free energyConstitutive
between the basalActivation
“on” and “off” states, such that the “on” state structure becomes
Below:
Mutation-Induced
the low energy state (meaning becomes the energetically favored state even in the absence of hormone). This is illustrated
below.
K basal > 1
“off” signaling state
K activation >>> 1
“on” signaling state
“on” signaling state
In this case, the signaling active form of the receptor predominates even in the absence of its cognate hormone. This is
inappropriate. The unstimulated signaling from such “constitutively active” mutant receptors can cause disease.
Human Vasopressin V2 Receptor and NSIAD
HN
2
P
E
L
D
R
D
P
L
L
39 A E
L
I
50 A
L
L
V
V
S
L
V
A
cytosol
L
S S
N
S P
L
S
P
L
T T
S A M L M
1
S
20
P R
R
C
G
F
A E
P
300
E A P
A
200
W
P
192 W
E V
N
G
L
G
C
D
D
R
R
D
G
E
A 112
R
W
D
Q
L
T
S
G
R
A
A
C
180
T
R
V G
100
A
F
T
A
A
A
L 289
V
W
Y
K
I
P
A
K
F
V
Q
F
A
W
R
L
Y
L
L
T
V
V
L
F
Q
W
L
Q
309
P
209
Q
F
I
L
P
L
L
A
M 120
S
L
V
P
A
A
L
L M
M
V
171
G
L L
L
F
W
Q
M
V
92
L
A
L
S
L
F
C
S
Y
A
V
S
V
F
L
A
S
Y
319
A
W
A
N
F
V
A
S
P
V
279
A
S
V
V
L
T
M
V
T
L V
L
D
I
A
131
I
A
V 160
I
G
N
V
L
A
L
W
P
A
L
M
R P
C
A 220
81
T
I
T
N
N
L
H
C
L
M
R
Y
Q
A
G
H W
G
D
V
V L
S
L
I
A
I
R
269
T
F
A 60
K
H
F
F
V
R
A
S
G
H
R
230
V
S
A
A
A
140
S
I
E
330
R
G
I
A
P A
S
I
R
W
H 150
V H
C
G
R
A H
260
R
S
H
A
R
P
Y
E
E D Q
G
70
G
R G
M
L
P
A
P
V
L
E R
S
S
S
P
P
C
G T R
P
T
G
G
T
240
R
G
360
G
R
R R
250
30
T
Q
H G P
10
S
V A
F
P
ectoplasm
V
S
L
E
S
S
G
C
L
L 340
C
A
R
S
G
R
L
R
S
350
P P
T
K
A
L
S
D T
S 370
S
Change of arginine-137 to
A S S
either cysteine or leucine
causes NSIAD: Neprhogenic
Syndrome of Inappropriate
Antidiuresis. It has been shown that these mutations result in constitutive activation of
the receptor, such that signaling occurs at all times.
Nephrogenic Syndrome of Inappropriate Anti-Diuresis (NSIAD)
A recently discovered disorder (May 5 2005 NEJM, 2 baby boys).
Very similar to the Syndrome of Inappropriate Anti-Diuretic Hormone
Secretion (SIADH). This disorder is caused by higher-than-usual levels of
vasopressin, the hormone that triggers vasopressin V2 receptor signaling.
Phenotype is opposite of diabetes insipidus: inappropriate retention of water,
resulting in hypoosmolar blood (specifically, low blood sodium) and overly
concentrated urine. One patient suffered seizures.
Patients are babies, so can’t be treated by restricting liquid intake. Instead,
urea was administered orally– an agent that increases osmolarity and thereby
triggers the formation of higher volumes of urine and fluid reduction. Babies
now OK (so far).
Vasopressin levels normal in these babies. Cause of NSIAD is mutations
of Arg137 of the vasopressin V2 receptor that make it constitutively active.
Recall that turning on the V2R turns on water absorption into the body,
leading to concentrated urine.
When are systems not at equilibrium?
For:
R + L
RL
Keq
If
[RL]
≠ Keq
[R]
.
[L]
Then, the system is not at equilibrium.
Potential (“driving force”) energy towards equilibrium:
Examples of non-equilibrium conditions:
Ligand binding after mixing.
Metabolic pathways.
Nascent proteins.
Energetically-trapped protein structures.
Enzyme reaction following mixing.
ΔG (0 at eq.)
What Are the Molecular Determinants of DGo?
DGo = DHo - TDSo
H: enthalpy. Negative is favorable.
T: temperature
S: entropy: degree of disorder. Positive is favorable.
Organizing anything involves a loss of entropy:
Creation of a concentration gradient
Connecting molecules together (whether covalent or not).
Folding a protein.
Binding of a flexible drug molecule to a rigid protein site.
Solvation.
The Hydrophobic Effect.
free alkyl
chains in water
aggregation
genesis of oil droplet
Interactions That Are Primarily Electrostatic
Van der Waals interactions
All atoms undergo VDW interactions.
What counts is intimate molecular
packing.
Repulsive VDW forces are basis
for “steric hindrance”
Ionic Interactions
+
d
q1 x q2
Energy Between Two Ions in Solution =
kxexd
q: charges of the ions
e: dielectric constant of solution between the ions
78 for water
2 for hydrocarbons
k: constant (get from a physics book)
Bond Dipoles
Helix Dipole
partial negative charge
partial
negative
charge
C-terminus
C=O
H-O
H-N
N-terminus
partial
positive
charge
partial
negative
charge
C-O
partial positive charge
H
net dipole
(dipole
moment)
of water
H
partial
positive
charge
when salt dissolves, salt bridges
beween Na+ and Cl- are replaced by
energetically favorable ion-water
electrostatic interactions
Hydrogen Bonding
H-Bond Donors: H-N H-O H-S
not C-H
:
H-Bond Acceptors: N:
O:
Some Terminology
Hydrophobic (“hates water”)
= Nonpolar = Apolar
Hydrophilic (“loves water”)
= Polar
•Unfavorable for polar molecule to be in a hydrophobic environment
•Hydrophobic molecules prefer to be in hydrophobic environment
•Water is a polar solvent
•Membrane bilayers have hydrophobic interiors
How much energy are these various interactions usually
worth in aqueous solution?
H-Bond and salt bridges: -1 to -5 kCal/mol
Loss of 1 degree of conformational entropy when rotation around
a single bond is frozen out: +1 kCal/mol
Hydrophobic Effect: transfer of 1 hydrocarbon –CH2- or –CH3 from
water to hydrocarbon phase: -0.9 to -2.1 kCal/mol
How Different Contributions to Molecular Energetics Can Create Order:
Formation of Lipid Bilayers, the Critical Structural Unit of All Biological Membranes
polar
head group
apolar tail
(acyl chains)
A bilayered lipid vesicle-- also called a liposome. By forming
sealed vesicles, the remaining hydrocarbon tails are shielded from water.