CHEM 4611
Survey of Biochemistry
What is biochemistry?
A
B
What things might biochemists be interested in?
This course is a survey course, but we will still talk about some
things in detail. It is a survey course because there are some
aspects of biochemistry we will not talk about and in others
where we will only cover them superficially.
Outline
Acids/Bases
Equilibriums, Ka, pKa, pH
Buffers
Acids, Bases and Equilibrium
Bronsted acids: donate protons.
Bronsted bases: accept protons.
1. NH4+ + H2O
NH3 + H3O+
2. NH3 + H3O+
NH4+ + H2O
So in fact: NH4+ + H2O
NH3 + H3O+
Equilibrium constant (Keq):
[Products]/[reactants]
[NH3][H3O+]/ [H2O][NH4+]
For acids and bases, we can take out the water and this is called the acid dissociation constant
(Ka).
Ka = [H+][NH3]/[NH4+]. The general form is: Ka = [H+][A-]/[HA]
Water, pH, and pKa
H2O
H+ + OHFor water K = [H+][OH-]/[H2O]
Kw = [H+][OH-] at 25°C is 1X10-14.
The H+ concentration in water (1X10-7M) is considered neutral.
If H+ is > 1X10-7 M the solution is acidic
If H+ < 1X10-7 M the solution is basic
pH = -log[H+]
pKa =
Buffers
• Definition
• Biological relevance
• How do they function?
pKa
HA
H + + A-
The dissociation constant is defined as Ka= [H+][A-]/[HA]
Ka can be expressed as pKa= -logKa. It indicates the ability of an
ionizable group to give up a proton. The lower the pKa. The
more easily the proton is given up. Acids have low pKa.
From the dissociation constant, you can derive Henderson –
Hasselbalch eqn:
pH= pKa + log[A-]/[HA]
Titrations and Changes in pH
Buffers Continued
Establishes an
equilibrium between
buffer’s components
Follows Le Chatelier’s
principle
Equilibrium shifts in
the direction that
relieves the stress
Figure 3.19 Titration of Acetic Acid
with NaOH
Changes in the Buffer Components
• Addition of acid (drops of HCl
not NaOH) eventually
converts all of the acetate to
acetic acid.
• Once the acetate is gone, the
pH drops sharply
Changes in the Buffer Components
Physiological Buffers
Buffers adapted to solve specific physiological
problems within the body
Bicarbonate Buffer
One of the most important buffers in the blood
CO2 + H2O
H+ + HCO3- (HCO3- is bicarbonate):
This is a reversible reaction.
Buffering Range is Dependent on the pKa
Weak acids
Weak acids act as a good buffer at pHs +/one from their pKa. Acetic acid has a low pKa
and is a good buffer at low pHs. Ammonium
has a high pKa and is a good buffer at high
pHs. But even acetic acid is a weak acid. HCl
is a strong acid.
PROBLEM:
Given: pH = pKa + log([A-]/[HA]) and the pKa of acetic acid is 4.76
How much acetic acid and sodium acetate is necessary to make a buffer solution of pH 5.5 and
0.05 M (acetic acid + acetate)?
We’ll say x is the concentration of the acetate anion (A-); total acetate/acetic acid is 0.05 M;
0.05 = [HA] + [A-]
0.05 – [A-] = [HA]
acetic acid (HA) is 0.05-x.
5.5 = 4.76 + log([x]/[0.05-x])
5.5-4.76 = log([x]/[0.05-x])
0.74 = log([x]/[0.05-x])
1X100.74 = [x]/[0.05-x]
5.49 = [x]/[0.05-x]
5.49*(0.05-x) = x
0.274 - 5.49x = x
0.274 = x + 5.49x
0.274 = 6.49x
0.274/6.49 = x
0.042 M = x = [acetate anion]
Remember:
We’ll say x is the concentration of the acetate anion (A-); total
acetate/acetic acid is 0.05 M; acetic acid (HA) is 0.05-x.
5.5 = 4.76 + log([x]/[0.05-x])
[acetate anion] = 0.042 M
[total acetate/acetic acid] = [acetate anion]+[acetic acid]
0.05 M = 0.042 M + [acetic acid]
0.05 M – 0.042 M = [acetic acid]
0.008 M = [acetic acid]
Does this make sense?
PROBLEM part II:
5.5 = 4.76 + log[A-]/[HA]
5.5 = 4.76 + log([x]/[0.05-x])
X= 0.042 M
0.05-x = 0.008 M
What happens if I double the [A-] and [HA]?
A.
B.
C.
D.
E.
F.
G.
H.
I.
The pH of the solution goes up
The pKa of the solution goes up
The pH of acetic acid goes up
The pKa of acetic acid goes up
The pH of the solution goes down
The pKa of acetic acid goes down
The pH of acetic acid goes down
The pKa of the solution goes down
None of the above
Why would a biochemist use a different [buffer]?
Your Turn…..
Buffers
MES
MOPS
Tris
CHES
CAPS
pKa
6.16
7.28
8.2
9.55
10.56
What would you use to make a buffer at a pH of 7.2? What would the [HA] and [A-] if the
[total buffer] is 100 mM?
Polyprotic Acids
Phosphate has multiple ionizable groups. Each of these groups has a separate pKa.
H3PO4 2.15; H2PO4- 6.82; HPO4-2 12.38
When making a buffer with phosphate, which pKa’s are a concern?
It depends on the pH of the buffer. The pKa of the acid (HA) in the buffer should be
used.
In general, when dealing with polyprotic molecules if the pKa’s differ by at least three units, pKa’s that are not
close to the “working” pH can be ignored. For example, at pH 7.8, we can ignore the H3PO4 because the
concentration of H3PO4 is essentially zero at pH 7.8. At a pH of 6.7, we can ignore the PO4-3 for the same reason.
This same reasoning is true for many biological molecules. For example, Histidine has 3 pKa’s (1.8, 6.0, and
9.3).
If the pKa’s are not far enough apart, things become much more complicated, and it is hard to determine the pKa’s
of individual groups.
Phosphate Titration
Titration of
Phosphoric
Acid with
NaOH
Weak Acids with Multiple Ionizable Groups
Each ionizable group can have its own pKa
Protons are released in a stepwise fashion
Biological Buffers
Physiological Buffers
Buffers adapted to solve specific physiological
problems within the body
Bicarbonate Buffer
One of the most important buffers in the blood
CO2 + H2O
H+ + HCO3- (HCO3- is bicarbonate):
This is a reversible reaction
Phosphate and phosphate containing molecules act as an important part
of a buffering system inside of many cells and organisms too.
Your Turn…..
A solution contains phosphate and is at a very high pH. What happens to the pH and the
phosphate as you slowly add in a strong acid? (Can you draw a graph of acid added vs. pH?)
Humans and Changes in pH
a blood pH of 7.2 ≤ is acidosis & a blood pH of 7.6 ≥ is alkalosis
Diabetic acidosis (also called diabetic ketoacidosis and DKA) develops when ketone bodies
accumulate during uncontrolled diabetes.
Alkalosis occurs when [bicarbonate] increases forcing the equilibrium to remove protons from
blood causing blood pH to rise. So pH becomes alkaline leading to vomiting, nausea, headache.
Respiratory alkalosis can be induced by excessive exhalation of carbon dioxide from lungs too
quickly causing too great a loss of H+ from the large reservoir (e.g. hyperventilation).
Biochemistry and Buffers
Many important biomolecules are affected by changes in pH. To ensure, constant pH in most biochemistry experiments and
methods buffers are used.
Different molecules are used depending on the desired pH (sometimes biochemists actually want non-native pH to understand the
role of the pH in processes) for buffers in vitro.
Examples of commonly used buffers:
Phosphate (e.g. H3PO4).
Acetate/Acetic acid
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)
Tris(hydroxymethyl)aminomethane (TRIS)
(unprotonated)
carboxylic acid
Glycine
O
O
amine
NH3
What do biochemists call glycine?
Outline
1. Structure of water
2. Forces that hold molecules/atoms together (interactions)
3. Introduction to amino acids (structure, pKa, and chirality)
4. Introduction to peptides and proteins (peptide bonds, N
and C-terminal)
The Structure of Water
Figure 3.3 Charges on a Water Molecule
Figure 3.4 Water Molecule
Polarity
Bonds between oxygen and hydrogen are polar
The electrons are not shared equally and there is a dipole.
Water has a dipole because the positive and negative
charges are separate
Polar molecules are generally
hydrophilic and interact well with
and dissolve in water and other
polar solvents.
Molecules that don’t have a
dipole are non-polar, generally
hydrophobic and don’t interact
with water well.
H bonds
An electron-deficient hydrogen of one water is
attracted to the unshared electrons of water
forming a hydrogen bond
Can occur with oxygen, nitrogen, and
fluorine
Has electrostatic (i.e., opposite charges)
components and covalent (i.e., electron
sharing) characteristics, but it is not a
covalent bond.
H bonds have preferred directionality and
distance depending on the groups involved.
What other types of molecules can be involved in H bonds?
How else can molecules/atoms interact?
More H bonds
D---H :A
Donor is the atom covalently attached to H,
acceptor less tightly associated with H
Types of Interactions
Interactions/bonds
Noncovalent interactions are electrostatic
Weak individually, but play vital role in biomolecules
because of cumulative effects
Interactions Between Charged Groups
Biochemists will talk about salt bridges and not ionic bonds because true ionic bonds
don’t normally occur in biochemistry. These interactions are driven by electrostatic
interactions but so are other types of interactions like H-bonds (partial positive H
interacts with a partial negative atom).
Lys
H
N+
H
Electrostatic force is given by Coulomb's
Law:
F = q 1 q2 / r 2 D
r = distance between the two charges q1
and q2.
Water has a large dielectric constant
(~80). If water is present,
it will weaken the force of attraction
between two charges, q1 and q2.
F is the force that measures the strength
of the interaction. If F is close to 0, then
the interaction is weak.
H
-O
O
Asp
Van der Waals Interactions
(A) Dipole-Dipole interactions (but not H-bonds or salt
bridges)
Occur between neutral,
permanent, and/or induced
dipoles
There are three types (A, B,
and C).
(B) Dipole-induced dipole interactions: If something with a dipole is brought near something without
a dipole, the electrons in the group without a dipole will move accordingly creating a dipole.
(C) Induced dipole-induced dipole interactions: The two CH3 don’t have dipoles on their own, but if
you force them to be close to each other, they will take on a dipole.
Van der Waals Forces Distance is Important
δ-
repulsion
attraction
δ+
Hydrophobic Effect
Oil (hydrophobic) and water don’t mix well. In water, oil molecules will come together to
avoid interacting with water. Why?
Many biomolecules are amphipathic. What happens?
Your Turn…..
Below is the dominant form of an amino acid called threonine at neutral pHs This amino acid
can be involved in H-bonds, van der Waals interactions, and salt bridges. What part of the
molecule could be involved in what interaction and what type of group/molecule would also
be the other part of that interaction?
Thermodynamics
A favorable process will have a negative ∆G.
∆G = ∆H - T*∆S
The ∆G for the lifting the barrier likely increases the entropy, has a negative
delta G, and is favorable.
Mixing generally increases entropy (S) so is thermodynamically more
favorable. You know less about the individual components if the system is
mixed. Things move more when mixing happens, which increases entropy.
More movement is generally more favorable.
Study Points
•
•
•
•
•
Be able to describe the bonds/interactions discussed: electrostatic interactions,
salt bridges, Van der Waals and H bonds.
Know the structure and properties of water
Understand how buffers work and how they relate to biological systems
Be able to manipulate and solve problems using the Henderson-Hasselbalch
equation
Be familiar with the following key terms: covalent bond, electrostatic
interaction, salt bridge, hydrogen bond, van der Waals interaction, hydrophobic
effect, entropy, enthalpy, free energy, pH, pKa, buffer
What’s coming:
•
•
•
•
•
Amino Acids and their structures and properties
Peptide bonds
Polypeptide chains
A little about DNA, evolution, and sequence alignments
Protein structure and folding
Amino Acids
amine
alpha-C
R group or
sidechain
unprotonated
carboxylic acid
(carboxylate
group)
Have central (a) carbon, an amino group,
carboxylate group, hydrogen atom, and an R
group/side chain (proline is the exception)
At pH 7, the carboxyl group is in its
conjugate base form (-COO-) while the amino
group is its conjugate acid form (-NH3+);
therefore, it is amphoteric (see notes for
comments. Notes will be in the slides through
out this handout).
R group or
O
sidechain
O
amine
CH
3
O
O
NH3
Gly
R is H.
NH3
Ala
R is CH3
Amino Acids Protein/Polypeptide Chain Structures
Primary structure:
The sequence of amino acids that make up a polypeptide chain, frequently given
by the 1 letter amino acid code e.g. (N-term) DASFGAELGMDEWAS (C-term).
The amino acids are the monomers and polymerize to make polypeptide chains
local structural units; does not include complex structures involving interactions
Secondary structure: between side chains (R groups). Related to the local structure of the backbone,
but side chain interactions are involved in secondary structure formation but
generally aren’t considered part of the secondary structure.
Tertiary structure:
The complete structure of a single polypeptide chain; the secondary structures are
together (all intramolecular interactions). Where are all the atoms in the
polypeptide chain.
Quaternary structure: Structure of complexes between more than one polypeptide chain (intermolecular
interactions)
In a protein, all 4 levels of structures exist. In the tertiary structure, we can still think about the amino acids
that make it up, which is its primary structure. The different levels of structures give different levels of
information. Primary structure is just what amino acids are in the polypeptide chain. The different levels
don’t represent an order in which events happen.
Chirality
With four different groups attached to a carbon, a-amino acids are chiral in many cases
(handedness)
Generally, L amino acids are used to make polypeptide chains in biological systems, but D amino
acids do exist and are sometimes used in biological systems for other things.
CORN Clockwise for L With the H Towards You
Gly has H as R and so isn’t
chiral. Of the 20 standard
amino acids, Gly is the
only one that isn’t chiral.
CORN
Some amino acids have more than one chiral centers
The difference between LThr and L-allo-Thr is the
chirality of a C in the
sidechain.
We will only really worry
about L and D, which deal
with the alpha-C.
L-Thr
Chiral C in
the
sidechain.
H
L-allo-Thr
COOCa
+ HN
C
H
3
OH
HO
COOCa
+ HN
3
C
H
C
CH3
C
H
CH3
How many forms of Thr are there?
For The 20 Standard Amino Acids L Are Equivalent to S, Except for Cys
H is going away from you.
Zwitterions
Increasing pH (adding base)
Dipolar ion
or zwitterion
What about side chains?
• There are 20 side chains (thus 20 amino acids) from which polypeptide chains from all branches of life
are constructed. These are called the 20 standard amino acids.
• They have different sizes, shapes, charges, hydrophobicity, H bonding abilities and chemical reactivities.
• You will need to know the 20 amino acids. Structure (including stereochemistry), three letter codes,
and the pKas (Figure 5.2 and Tables 5.1 and 5.2 of your book). For the N and C-term, we will use ~2 and
~9 for the pKa values and not the values in the table 5.2 in the book. You will have to know the 7 pKR
values in table 5.2 and what those values mean with respect to the structure of those amino acids. The
help file on BlackBoard for amino acids pKa also gives useful information on the pKas and protonation
states. The structures are on the next slide.
• These are things you will have to know all semester for (every quiz and test). You should understand
what these values and structures mean in terms of interactions and what the amino acids can do (e.g.
act as an acid or base, be involved in H-bond, etc.)
• The pKa of HCl is -6.3. The pKa value of H2O is 14.
H+
Peptide Bonds
alpha-C 2
alpha-C 1
alpha-C 2
alpha-C 1
Another amino acid
could be added
here!
Peptide bonds are formed by making a bond between the N attached the alpha-C of one amino
acid and the C of the carboxyl group attached to the alpha-C of another amino acid. The
reaction shown is thermodynamically unfavorable under biological conditions and isn’t how the
reaction actually happens, but the idea of losing a water is a useful way to remember how to
draw a peptide bond.
The result above contains two amino acids and so is a dipeptide.
What functional group is a peptide bond? What properties does it have?
Limits on Polypeptide Structures
Limits on Polypeptide Structures
The peptide bond is rigid and flat
C-N bonds between two amino
acids are shorter than other C-N
bonds
The have partial double-bond
characteristics because they
are resonance hybrids
Because of the rigidity, one-third
of the bonds in a polypeptide
backbone cannot rotate freely
Limits the number of
conformational possibilities
Two configurations for the planar peptide bond
R-group (or side chain)
*
*
R-group (or side chain)
Trans – the a carbons
(*)are on opposite
sides of the peptide
bond
*
*
R-group (or side chain)
Cis – the a carbons are
on the same side of
the peptide bond
Which is the preferred conformation?
Proline Cis vs. Trans
Peptide
bond
L-proline
• About 10 percent of bonds with
Pro as the C-terminal amino acid
are found in cis, based on known
polypeptide structures.
• Other cis amino acids do occur
in polypeptide chains, but they
are even more rare.
• Isomerization of Pro from cis to
trans can be slow.
L-proline
Peptide
bond
Peptides Have Directionality
The Phe and Leu have
a peptide bond that is
in the trans
conformation. The
sidechains are on
opposite sides.
Another amino acid
could be added
here!
•There is polarity to polypeptide chains
•The amino end is the beginning of the polypeptide chain
What is the charge on the L-Tyr Gly Gly L-Phe L-Leu pentapeptide at a pH of 11?
Your Turn…..
Draw the dominant form L-Val L-Asp at a pH of 7?
Polypeptide Chain Backbone
What is the polypeptide chain “backbone”/ main chain?
This is the constant repeating part. It is the non-sidechain/R groups of the
polypeptide chain. The atoms in black are the backbone atoms.
alpha-C 3
alpha-C 1
alpha-C 2
alpha-C 5
alpha-C 4
The stretch above shows 5 amino acids. Each has an alpha-C and a sidechain
(R). The amino acids are L amino acids. The peptide bonds are in the trans
conformation.
Polypeptide Chains Can Contain Other Covalent Bonds
Cystine contains a disulfide bond.
DNA and Polypeptide Chains
Different polypeptides have different structures due to different sequences (i.e. they have
different amino acids in different orders, the order of the amino acids in a polypeptide chain is
called its primary structure).
Different amino acids can undergo different interactions based on the sidechains of the amino
acid. Some (e.g. Ser) can be involved in H-bonds. Others can’t but can be involved in van Der
Waals interactions. Etc.
A central idea in biochemistry is that the sequence of a polypeptide is related to its structure and
structure is related to function. Sequence dictates structure, and structure dictates function.
The sequence of a polypeptide chain in biological systems is dictated by the DNA sequence.
Like polypeptide chains, DNA is a polymer made up of monomers. There are only 4 DNA
monomers, which are abbreviated A, G, C, and T. The order of these monomers in the DNA
molecules dictate the order of amino acids used to make polypeptide chains.
DNA and Mutations
DNA is not a static structure. For organisms to grow and reproduce, cells must divide. In most
cases, when cells divide the DNA in the cell must be copied so that each cell keeps the same DNA.
Sometimes during this process of replicating the DNA an error is made, and the copied DNA is not
the same as the original DNA molecule. This change would be called a mutation. Mutations can
affect the sequence of a polypeptide chain.
If the sequence of a polypeptide chain is affected, the structure and function of the polypeptide
chain can also be affected.
DNA inside of cells also sometimes comes into contact with reactive molecules.
Reactive molecules can chemically alter the structure of the DNA and therefore cause a mutation.
Any mutation might result in some polypeptide chain(s) having a different sequence that then
might affect its structure and function.
Polypeptides and Evolution
Mutations in DNA can result in a change in the primary sequence of a polypeptide.
It is not believed that organisms can specifically cause mutations in specific parts of the DNA to cause a
specific change in a polypeptide chains sequence.
Mutations can result in the change of an amino acid or insertion or deletion (indel) of amino acid(s). A,
G, C, and Ts can be removed or added to the DNA sequence causing the same effect in the polypeptide
chain.
Mutations can have a negative effect, a positive effect, or a neutral effect.
For a mutation to have a positive effect, the organism containing the mutation must
leave more viable off-spring (they can reproduce).
Positive mutations will be conserved by evolution.
Negative mutations will be lost.
Closely related organisms (E. coli and S. typhirium) will have few or no differences
between many of their polypeptides.
The polypeptides with the same function in different organisms that are well evolved (they first arose long
ago, and their function has changed very little in different organisms) will have few differences in their
sequences.
Amino Acid Changes
Due to mutations accumulated over time, different organisms have DNA molecules with different
sequences and so make polypeptide chains with different sequences.
But in many cases, some of these polypeptide chains retain very similar structures and similar or identical
functions. Our DNA, and the resulting polypeptide chains, are different than those in dogs, but we share
DNA from a common mammalian ancestor that allows us to make similar polypeptide chains that result in
similar biological structures (e.g. fur/hair, toenails (these things are polypeptide chains)).
A functionally important position in a polypeptide is less likely to change as a result of evolution (most
changes at that position will be negative).
In comparing polypeptide chains between organisms, positions in a polypeptide can be:
Invariant
Conservatively substituted (e.g. Asp to Glu)
Hypervariant (many different kinds amino acids are acceptable at that position).
Even at hypervariant positions some amino acids may have a negative effect.
Amino Acid Changes
G (Gly) is the first, the N-terminal, amino acid in both polypeptide chains.
Q (Gln) is the last, the C-terminal, amino acid in both polypeptide chains.
The purple indicates one polypeptide chain. Each amino acid is represented by one letter (one letter
code). The purple then represents one molecule where each amino acid is connected to the next through
a peptide bond. The blue is another polypeptide chain (another molecule) where each amino acids in
connected to the next through a peptide bond.
All of the amino acids in these two polypeptide chains are the same except for the highlighted position.
The sequence of these polypeptide chains would be dictated by different DNA molecules. Those two DNA
molecules would also have similar sequences, which results in the polypeptide chains having similar (and
nearly identical) sequences.
Sequence Alignments
We want to line up amino acids
that are in similar structural
positions and so have similar
functions without having to
actually determine the
structures.
Sequence Alignment and Indels
The white is the backbone of the hemoglobin alpha polypeptide chain. The red and green structure is the
backbone of the myoglobin polypeptide chain. The green is the area of the myoglobin that corresponds
to the indel based on the sequence alignment.
A Multiple Sequence Alignment.
hemoglobin_alpha_chain -VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFP
hemoglobin_beta_chain VHLTPEEKSAVTALWGKVN--VDEVGGEALGRLLVVYPWTQRFFE
myoglobin
-GLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFD
hemoglobin_alpha_chain HF------DLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSAL
hemoglobin_beta_chain SFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATL
myoglobin
RFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEIKPL
hemoglobin_alpha_chain SDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKF
hemoglobin_beta_chain SELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKV
myoglobin
AQSHATKHKIPVKYLEFISEAIIQVLQSKHPGDFGADAQGAMNKA
hemoglobin_alpha_chain LASVSTVLTSKYR-----hemoglobin_beta_chain VAGVANALAHKYH–----myoglobin
LELFRKDMASNYKELGFQG
Sequence Alignments as Tools to Treat Diseases
Today, we know the sequences of many polypeptide chains from many different organisms.
We can compare the sequences and determine which ones are likely related to one another.
We can identify amino acids that are conserved between these likely related polypeptide chains using tools
like sequence alignments. Then we can determine what amino acids that are likely important for the
structure and function of polypeptide chains.
Sequence alignments are important tools in studying how organisms are different and similar and
identifying things that make polypeptide chains similar (e.g. have similar structures and functions) and
different.
These tools were used to compare the polypeptide chains made by the Covid-19 virus and other viruses
that make similar polypeptide chains. This information could then be used to identify molecules that are
being used against other viruses to understand if they would be effective against the Covid-19 virus or why
they wouldn’t be effective.
In a solution at a pH of 7 containing L-Asp L-Ser, the dominant charge on the dipeptides will be:
A.
B.
C.
D.
E.
-2
-1
0
+1
+2
Learning objectives- you should understand:
• What an amino acid, a peptide, and a peptide bond is.
• You should understand the chirality of amino acids and how the charge
on an amino acid is related to the pKa of the groups on the amino acid
and the pH.
• terminology associated with chirality, peptides, the peptide bond, and
charges of amino acids/peptides
• The different levels of protein
• What sequence alignments are, how they are related to the sequence
and function of proteins, what they are used for, and some
terminology associated with them.
• How sequence alignments are related to evolution and some basic ideas
related to DNA and evolution and the associated terminology.
What’s coming:
Details on the different levels of structure
Primary
Secondary
helix and beta sheet
Tertiary
domains
Quaternary
subunits
What level of structure?
MPPKNNPKQHANVNGNHKLDSRPSNETGRPKREENKADDVVVPAGPTIKAQITNASKTSSGKTFLEALRTNV
TKQTVRVEKPRTPEPPEVAVPEPEVPVAEEPTVEAVVAPVEIPPTPVEESPVVEKQPTPTPKPQQPPKPQPTVTL
PESNGNFSWANDDDYTFSSDITPQHPVVGPPQAKPVTYIVTYPAEVVEAHSKGPRVVFEAPASHQRIDSAMAE
LEKRANLYNERHLFEECRKQREIEMNEKHARLLAQERQLNDQSDGLQRERSRLVEQQQQLQQQQQQQQQQ
QMIQQQPAPLRQAPSSLPVTSQTQLPPPPQPPQHVGNNYTYQTGTQDGMPWGGRGRNDHWTPNAGVM
VPPYDHNHYYSGGHYLMQRNQNFGDRGVDVPPHMGRGNAMRGGPHMGGFSAGRPMGGADGGVNQM
FGQRNSGGYMGGPALPNRW
N-terminal vs. C-terminal
The amino acids of the polypeptide chains are held together by peptide bonds. Between each
amino acid, represented by a letter, is a peptide bond.
Secondary Structure
Secondary Structure: Polypeptide
secondary structure has a variety of
repeating structures
Most common include the a-helix and bpleated sheet
Both structures are stabilized by
hydrogen bonding between the carbonyl
and the N-H groups of the polypeptide’s
backbone
There are other types of helixes and
secondary structures
How what variables would be useful in
describing a helix?
Beta-pleated Sheet
The b-pleated sheets form when two or more
polypeptide chain segments line up, side by side
Fatty acid binding protein
An example of a b-sheet
rich protein
Turns and Bends
Proteins often have compact globular shapes. How?
Reverse turn/b-turn/hairpin turn
Beta-pleated Sheet II
Turns and Bends
Loops (non-repeating secondary structures)
Tertiary Structure
Myoglobin carries stores oxygen
in cells.
153 amino acids, very compact.
The globin fold.
myoglobin
Myoglobin is a globular proteins. Globular proteins have
a compact structure.
Interactions in Tertiary Structures
Interactions that stabilize tertiary structure are
hydrophobic interactions, salt bridges, hydrogen
bonds, covalent bonds, and hydration
Protein Domains
Domains are independent folding units that sometimes maintain some
function independent of the rest of the polypeptide chain. The interactions
between domains in a protein tend to rather modest.
Some Evolution Vocabulary
Homologs
Orthologs
Paralogs
Your Turn…..
Describe what you see? How does what you see likely differ from reality?
Convergent Evolution
Both Chymotrypsin and subtilisin are proteins that cleave peptide bonds in other proteins, and
they use Ser, Asp, and His that are part of their structure to do so, but they are not homologs.
Structure is Conserved?
Quaternary Structure
Quaternary Structure: What is it?
• For example, a dimer (two polypeptide chains – each chain is
called a subunit)
• Can be identical or
different subunits
• Can be multiple subunits
Types of Quaternary Structure
A protein maybe made up of multiple polypeptide chains and are called oligomers.
A protein made of more than one of the same polypeptide chain is a homomer.
Proteins can also contain different polypeptide chains and are called heteromers.
Homodimers, heterodimers, homotrimer, heterotrimer, etc.
Heterotetramer: ab3, a2b2, a3b
Different proteins that can function independently also form complexes in vivo.
Protein-protein interactions are specific though. Unspecific interactions would result in clumping, aggregation,
and precipitation of proteins.
Protein-protein interactions, which includes formation of non-monomer quaternary structure, is driven by
different types of interactions in different cases and includes essentially all the types of interactions we have
talked about (covalent (disulfide bonds), H-bonds, hydrophobic/van der Waals, and salt bridges). Amino acid
side chains and the peptide backbone groups between polypeptide chains interact when polypeptide chains
interact.
Quaternary Structure
4 subunits:
2 light chains
2 heavy chains
Covalent and noncovalent interactions
Reasons for common occurrence of multisubunit
proteins:
1. Synthesis of subunits may be more efficient
2. In supramolecular complexes replacement of
worn-out components can be handled more
effectively
3. Biological function may be regulated by complex
interactions of multiple subunits
Learning objectives- you should understand:
• The different levels of protein structure and examples of them and
how they are different and related
• Forces related to the formation of protein structures
• Basic terminology related to protein structures such as domains and
subunits.
• Some basics of evolution and vocabulary related to them, including the
meaning of orthologs, paralogs, homologs, and convergent evolution
Your Turn…..
A solution with water and NaCl is made. The NaCl is 10 mM. In the solution, the Na+ and ClA.
B.
C.
D.
Interact strongly and specifically with one another
Move around a lot and interact with lots of partners and the water molecules
Repel one another
Are involved in van der Waals interactions
What’s coming:
Protein folding
Protein folding landscapes
Hydrophobic collapse
How do Proteins Fold
In dilute solutions, many proteins can fold into their biological (native) structure on their own.
The peptide bond and the properties of the amino acids limit the structures that polypeptide chains can take
on.
Many structures are unfavorable (then system has a high Gibbs energy). E.g. for most polypeptide chains having
many peptide bonds in the cis conformation is unfavorable.
The hydrophobic effect is important. Hydrophobic amino acid sidechains interact. Water molecules no longer
must interact with them then, and the water molecules gain entropy.
In protein folding, the process of the hydrophobic amino acids coming together to interact is called hydrophobic
collapse.
The gain of entropy from the water molecules during hydrophobic collapse can drive protein folding and make
the delta G for the process negative. The Gibbs energy of the system goes down.
Protein Folding Landscapes
x
Y
(Gibbs energy)
a
.
.
b
.
c
z
The N (native) conformation results in the lowest Gibbs energy. The native structure is the biologically
relevant structure of the protein. The conformations that correspond to points a, b, and c all have the
same Gibbs energy. Most proteins don’t have protein folding landscapes that look like this.
Protein Folding Landscapes as a Funnel
x
c
a
Y
(Gibbs energy)
.
b
z
Most proteins don’t have protein folding landscapes that look like this either.
Realistic Protein Folding Landscape for Many Proteins
Example of unfolded protein
(high Gibbs energy)
x
Example of unfolded protein
(high Gibbs energy)
Y
(Gibbs energy)
z
N
Example of partially folded protein (molten
globule conformation). Hydrophobic
collapse and formation of secondary
structure have started to happen. They can
also be intermediates in the folding process.
Your Turn…..
Which is more favorable, two oppositely charged groups interacting on a surface of a protein all of the
time or two oppositely charged groups on a protein moving and interacting with other things at
different times?
A.
Water based solution with salts (e.g. KCl, phosphates, MgCl2, etc.)
Outside of oval is a polar protein surface in general
COOCOONH3+
NH3+
time
hydrophobic
hydrophobic
OR
B.
COOCOO-
NH3+
NH3+
time
hydrophobic
hydrophobic
Which is more favorable, (See the notes for more context)
Outside of oval is a polar protein
A.
surface in general
Water based solution with salts (e.g. KCl, phosphates, MgCl2, etc.)
+
NH3
COO-
hydrophobic
B.
COO-
A. The negatively charged carboxylate group is
surrounded by the hydrophobic core.
B. The positively charged amine is surrounded by
the hydrophobic core.
NH3+
hydrophobic
C.
COO- +
NH3
hydrophobic
C. The positively charged amine and
negatively charged carboxylate group are
surrounded by the hydrophobic core, but
they can interact with one another.
Fibrous Proteins
Things like nails, hair, collagen, and silk are fibrous proteins.
• Fibrous proteins have elongated structures
(fibers). They are not compact.
• An example of an alpha-helix based fibrous
protein. There are fibrous proteins that are
made mostly of beta-sheet (e.g. silk).
• They tend to be dominated by a single type of
secondary structure
• They normally are involved in structural roles.
Not all proteins are the same. Some proteins need to take on structures in the absence of water (e.g. hair, nails, etc.). Then
hydrophobic collapse can’t be a driving force for protein folding.
What’s coming:
•
•
ATP
Chaperones and connecting ATP hydrolysis to protein folding
ATP, The Energy Currency of the Cell
Adenosine triphosphate is a
nucleotide that plays an
extraordinarily important role in living
cells
Hydrolysis of ATP + H2O  ADP + Pi
provides free energy
ATP:
For now, we’ll only worry about the phosphates.
ATP Hydrolysis Drives Processes that Otherwise
would be Thermodynamically Unfavorable
Drives reactions of
several types:
1. Biosynthesis of
biomolecules
2. Active transport
across membranes
3. Mechanical work
such as muscle
contraction
Reactions involved in biosynthesis tend to be?
Chaperones
Sometimes proteins need help folding or can’t fold on their own.
Chaperones are proteins that help other proteins fold.
There are different types of chaperones. One type of chaperone binds to misfolded proteins, uses the
hydrolysis of ATP as energy to unfold the proteins, and then lets the misfolded protein fold on its own in a
protective cage.
These proteins are often called heat shock proteins (HSPs).
Phosphoryl Transfer Reactions
The tendency of ATP to undergo hydrolysis is an example of its phosphoryl group transfer
potential
ATP acts as energy currency, because it can carry phosphoryl groups from high-energy
compounds to low-energy compounds
ATP Hydrolysis
Several factors need to be considered to understand why ATP is so exergonic (i.e.
have such a low delta G):
1. At physiological pH, ATP has multiple negative charges
2. Because of resonance stabilization, the products of ATP hydrolysis are more
stable than resonance-restricted ATP
3. Hydrolysis products of ATP are more easily solvated
4. Increase in disorder with more molecules
Remember other molecules are higher energy phosphate donors than ATP. So
why use ATP for energy?
R
Learning objectives- you should understand:
• How proteins fold
• The energetics and importance of ATP and high energy phosphate
molecules.
Biochemistry in the Lab: Experimental
Techniques to Study Proteins
What’s coming: Protein techniques
1. Protein purification/preparative techniques
salting out, chromatography, etc.
Analytical techniques:
2. Visualizing proteins and separating proteins without purification.
Gel electrophoresis
3. Detecting a protein (is the protein there, and how much?)
ELISA and Western blot
4. Structural determination.
X-ray crystallography, NMR, and cryo-EM
Background
• Why do you want to purify proteins?
• Why is this relevant?
Assay example: Lactate dehydrogenase
NADH absorbs light
at 340nm, NAD
does not.
Can we use this to our
advantage?
NAD+
NADH
Protein Purification
Proteins have physical and chemical characteristics that are dependent
upon their sequence and structure.
These characteristics can be used to separate proteins from one another.
SizeSolubility-
Chromatography
Liquid chromatography is an important part of purifying proteins and other
biomolecules.
Buffer
solution
resin
column
Collection
tube
Ion Exchange Chromatography
CM is an example of a resin that can be used for
cationic proteins (cation exchange chromatograph).
DEAE is an example of a resin that can be used
with anionic proteins (anion exchange
chromatography)
What is the isoelectric point (pI) of a protein?
Size Exclusion/Gel Filtration Chromatography
Good for analyzing protein quaternary structure
affinity chromatography
You can manipulate the gene of interest to use with
affinity chromatography…
Gene of interest
DNA encoding for a “tag” that will be utilized in affinity chromatography
e.g. The His tag
binds well to Ni, which
can be used to make a
resin and then for
chromatography.
His Bound to Ni Resin
Sodium Dodecyl Sulfate (SDS)
Gel Electrophoresis
cathode
anode
SDS-PAGE: SDS-Polyacrylamide Gel
Electrophoresis
DENATURING CONDITIONS!
Gels can also be done with out SDS (native
gels).
•Gels made of polyacrylamide are used (you
can control the pore size)
•A reductant is often added to break disulfide
bonds.
•Gels are like a molecular sieve
After staining and destaining:
Putting this altogether To Purify a Protein
Lyse the cells
Cells in a solution
Centrifuge
Purification
step (e.g. IEC)
Cells lysate contains proteins and
other things (cell “debris”)
Assay the
collected
fractions
Subject fraction(s) with
activity to more
purification steps as
needed.
Run SDS-PAGE Abs.
on fraction(s)
with activity
time
Your Turn…..
Polypeptide chain
Molar mass
pI
A
50,000 grams/mole
9.2
B
49,000 grams/mole
5.5
C
30,000 grams/mole
9.1
How would you purify large amounts of protein A from a solution that contains A, B, and C?
Another type of Gel Electrophoresis: Isoelectric focusing
• Electrophoretic separation of proteins based on the amount of acidic
and basic residues present.
• When the net charge of the protein is zero, it will not migrate in an
electric field. NATIVE CONDITIONS
Decreasing pH
Proteins will migrate until they
reach the pH where their net
charge is zero
IEF: Proteins migrate based on their charge in a pH
gradient.
Antibodies
• Antibody (immunoglobulin/Ig): a protein produced in response to an
antigen
• Generally Specific and high affinity for antigen
• Antigens include protein, polysaccharides, peptides and nucleic acids
Four polypeptide
chains make up an
antibody (IgG in this
case)
Ribbon Diagram of Antibody Structure
Epitope
(or antigenic determinant): the portion of the target molecule
recognized by the antibody
ELISA
ELISA tests for the presence of an antigen
Antibody to
antigen of
interest
Add blood,
urine, etc.
that might
contain
antigen
A different
antibody to
the antigen
is added
Western Blot
Western Blots test for the presence of a polypeptide chain.
Y
YY Y
Y
Run
SDS-PAGE
Transfer the
polypeptide
chains to a
membrane
Wash and add
another
antibody that
can bind to
the first
Add an
antibody that
will bind to
the protein of
interest.
Y
YY Y
Which
polypeptide chain
is biggest?
Western blots use primary antibodies to bind to the protein of interest. A secondary
antibody is then used to detect the primary antibody. After the secondary antibody is
added, the membrane must be washed again to remove any unbound secondary antibody.
Y
A Positive Western Blot
In most cases, this requires two
antibodies. A primary antibody to
recognize your protein and a
secondary antibody for detection.
All of the polypeptide chains
show in blue in the SDS-PAGE are
present in the western blot. They
are just not being detected
because what is being detected is
a signal specific to the secondary
antibody.
Your Turn…..
A protein is known to cause one type of cancer, you’ve been charged with
determining if it is present in other types of cancer cells. What technique would be
useful to you?
A. affinity chromatography
B. SDS-PAGE
C. ELISA
D. Western blots
Protein Structures
1. 3-D structures of proteins and other important biological entities at atomic detail can be
deduced via X-ray crystalography and NMR.
2. X-ray crystallography only gives a static structure and not all proteins can be crystallized
(proteins do have to move to function).
3. NMR is most useful for smaller proteins (<40 Kd).
4. In some cases, (cryo)-electron microscopy (cryo-EM) has been can be used to determine
structures at atomic levels. Cryo-EM is heavily used for things that are large and don’t
crystalize well (e.g. large protein complexes).
5. The Protein Data Bank (PDB) is a database that stores protein structural data
(www.pdb.org).
Learning objectives- you should understand:
• The principles behind the purification methods we discussed (e.g.
salting out, affinity chromatography, size exclusion), how protein
purification works, and why it is important
• Gel electrophoresis, including different types of gel electrophoresis
and what it is used for.
• ELISAs and Western Blot analysis
• How antibodies work and some of the applications
• General information on analyzing proteins and determining the 3-D
atomic structures of proteins.