Protein structureandfunction 2018_fall3 (1)

Protein Structure and Function
Pedersen 2018 September 6 1
Steen E. Pedersen
spedersen@rossu.edu
Email for appointment.
Required Reading: Meisenberg Chapter 2.
There are good practice questions at the end of the chapter, and also posted on e-
college.
Some of them are not addressed in lecture but require reading the book to get the
answer.

Learning Objectives
1. Explain what determines the primary structure of a protein.
2. Explain the nature of the amino acids including the following concepts: chirality,
stereoisomers, and functional groups.
3. Given the 1 letter or 3 letter code for an amino acid, provide the full name for that amino acid.
4. Given the name or code for an amino acid, identify the functional group that is on its side
chain.
5. Given a pKa, determine at what pH the amino acid or chemical will buffer best.
6. Given a primary structure of a peptide, determine the net charge at physiological pH.
7. Given the genetic code table, translate a nucleic acid sequence into an amino acid
sequence.
8. Compare and contrast the kinds of bonds involved in secondary structure stability and
tertiary structure stability.
9. Explain the physiological role that the following proteins have in the body: hemoglobin,
myoglobin, collagen, superoxide dismutase.
10. Explain what an enzyme is and what is an enzyme’s function is in making life possible.

Coverage
• Bottoms up:
– Individual amino acids and their properties
– Peptide bond properties
– Genetic code
– Biological Protein Synthesis on Ribosomes
• Basics of protein structure
– Secondary structure: α-helices, β-sheets, and turns.
– Tertiary structure: Domains
– Quaternary structure: Assemblies
• Charge properties – pH
• Examples of Protein Function
• Examples of Post-translational Modifications

Amino acids are the building blocks ofproteins
• An α-amino acid consists of a central α carbon
atom, an amino group, a carboxylic acid
group, a hydrogen , and a distinctive R group.
• The R group is the side chain.
• Since four different groups are connected to
the tetrahedral α- carbon atom, they are chiral.
• The two mirror-image forms are called the L
isomer and the D isomer
• Only L amino acids are found in proteins
Key Points
1. Many biomolecules have Chirality: non-identical mirror images.
2. Amino acid used for protein synthesis are L isomers.
3. The D isomers of some amino acids alse exist in nature and may be used for special
purposes (e.g. making cells walls in bacteria).
4. Because proteins are made from L isomers of amino acids, they are also inherently
chiral.

Amino Acid Categories
• Amino Acids can be categorized
on the basis of their side chain
properties:
– Charge
– Hydrophobicity
– Polarity
– Aromaticity
– Size
• Some AAs have specific effects
on secondary structure: Pro, Gly
Amino acid chains – typically drawn Amino to Carboxy
• Backbone atoms include Nitrogen, alpha-carbon, and carboxylate group, with the
associated hydrogen.
• Side chains, also called R-groups, sprout from the alpha-carbon.
• These side-chain functional groups include alcohols, thiols, thioethers, carboxylic
acids, carboxamides, and a variety of basic groups.
• When combined in various sequences, this array of functional groups accounts for
the broad spectrum of protein function.
• For instance, the chemical reactivity associated with these groups is essential to the
function of enzymes, the proteins that catalyze specific chemical reactions in
biological systems.

• Small Amino Acids:
– Glycine, Gly, G
• Very flexible backbone
• Smallest AA
• Often found in turns and active
sites
– Alanine, Ala, A
• Single methyl group
• Moderately hydrophobic
• Proline, Pro, P
– Breaks or kinks α-helices
– Specialized structures
– Major constituent of collagen
– Modified (hydroxylated).
Small Amino Acids and Proline
Generally, the smaller amino acids are more flexible, because they have small R-
groups that don’t impede bond rotation around the alpha-carbon bonds. Thus, they
are important in special structures in the protein, such as active sites and turns and
random coils that connect more regular secondary structures.
Proline also is present in unusual secondary structures. The constraint of the
R-group self-bonding with the nitrogen, does not permit proline to form straight alpha-
helices; so it is rarely found in those structures.
• Gly is important in proteins that form twisted helices with other proteins (like
collagen) since it has a small R group which allows tight packaging of the
strands forming the helix.
• Pro tends to break the α-helix of protein secondary structure.

• Branched Chain Amino Acids:
– Valine, Val, V
– Leucine, Leu, L
– Isoleucine, Ile, I
• Hydrophobic –
– Typically found in hydrophobic
cores of proteins – structural.
Branched Chain and Hydrophobic Amino Acids
• Val, Leu, and Ile are called branched chain amino acids (BCAAs).
• The BCAAs are hydrophobic and are often in the core of proteins.
• Metabolically, when muscle protein breaks down to amino acids, the amino group
of a BCAA is given to pyruvate to make Ala and the carbons of the BCAA are sent
to the TCA cycle to extract energy for the muscles.

• Hydroxyl and Sulfur Amino Acids
– Serine, Ser, S
– Threonine, Thr, T
– Cysteine, Cys, C
– Methionine, Met, M
• Ser and Thr and Cys are polar
amino acids
– H-bonding, reactive sites, structural
• Cys is similar to Ser, but Sulfur
reactivity is a little different.
• Met is hydrophic; it can act as a
single methyl group donor.
Hydroxyl and Sulfur-containing Amino Acids
• Serine’s and Threonine’s hydroxyl groups are very reactive and can be
phosphorylated or glycosylated easily.
• They also participate in active sites to carry out covalent chemistry or
acid/base chemistry
• Methionine:
• The methyl group that is attached to the sulfur atom of methionine is able to
be given to other molecules that need a “1 carbon group” to grow larger. It
is a methyl group donor.
• Cysteine can form a “disulfide” bond with another cysteine. This is critical in protein
tertiary structure formation and sometimes in quaternary structure.
• Formation of cys-cys disulfide bond is an oxidation reaction. Reversing it is a
reduction:
cys-SH + cys-SH cys-S-S-cys + 2H (note – the Hydrogens are
incorporated into other molecules).
• Reducing agents specific for disulfides can reverse this, such as glutathione,
which is part of the intracellular redox system.

• Aromatics
– Phenylalanine, Phe, F
– Tyrosine, Tyr, Y
– Tryptophan, Trp, W
• Function
– Somewhat hydrophobic
– Stacking interaction with substrates
– Tyrosine phosphorylation
– Active sites
Aromatic Amino Acids
Aromatics are large, generally hydrophobic amino acids (but less hydrophobic than
branched chain amino acids).
• They play special roles in stabilizing the binding of aromatic rings (such as in ATP).
• Tyrosine is a precursor for many other compounds.
• Tyrosine is phosphorylated during growth factor receptor activation. A phosphate on
a tyrosine is a “docking” site for proteins with a “SH2 domain”.
• Phe can be converted to Tyr by a hydroxylation reaction.
• Trp can be converted to the B vitamin, niacin.
Note: Hydrophobicity is a continuous scale. The question of the relative hydrophobicity
of aromatics often arises (both from students and in research). Aromatics are not as
hydrophobic as the large aliphatic residues (Leu, Ile, Val), but is more hydrophobic any
of the polar residues. You can convince yourself of this by thinking of some of the
chemical equivalents of the side chains:
For Phe, this is methyl-benzene, which is immiscible with water, and quite
hydrophobic.
For Tyr, this is methyl-phenol. Phenol itself is not miscible with water, except to a small
extent.
So, aromatics tend to play a unique role, but are rarely on the surface, but appear in
the interior or at active sites or at interfaces between polar/nonpolar regions (eg,
lipid/aqueous interface).

• Acidic Residues
– Aspartate, Asp, D
– Glutamate, Glu, E
– Negatively charged
– Generally on the exterior to impart
solubility
– In active sites
– Protein modifications
• Acidic residue derivatives:
– Asparagine, Asn, N
– Glutamine, Gln, Q
– Generally polar, uncharged
– Can be reactive,
– Asn is the site of N-linked
glycosylation.
Acidic Amino Acids and their Amides
• Aspartate and Glutamate have carboxylic acid side chains. These are charged at
neutral pH.
• They are typically found on the exterior of proteins to help keep them soluble
in water.
• They can be modified or participate in reactions in enzyme active sites.
• They are very polar, because of the charge.
• Asparagine and glutamine are the amides of aspartate and glutamate.
• They are uncharged and polar.
• Asparagine is used for N-linked glycosylation of proteins.
• Glutamine is used as a nitrogen carrier in circulation, in addition to being a
protein constituent.

• Arg is part of the Urea cycle
• Lys – undergoes modification
• His – often in active sites, pK is
near neutral and can be readily
protonated
• Basic Residues
– Lysine, Lys, K
– Arginine, Arg, R
– Histidine, His, H
• Charged at neutral pH
• Typically found on the exterior to
render the protein soluble
Basic residues: Lysine, Arginine, Histidine.
Histidine can bind or release protons (act as a buffer) near physiological pH.

Amino acid
3 letter codes
1 letter codes
(take the time to know these)
Protein Structure and
Function
Amino Acids
You have to recognize the codes for the amino acids. This may be a bit painful, but its
part of your working knowledge.

Functional groups
• The functional groups of proteins are
usually found in their amino acid side
chains.
• Hydroxyl – Ser, Thr, Tyr
• All of these can be phosphorylated on their side
chains
• Methyl – Ala, Val, Ile, Leu, Met
• Met can donate its methyl group to other
molecules that need a carbon atom to make
bigger molecules
• Carboxy – Asp, Glu
• Amino – Lys
• Sulfhydryl – Cys
• Cys can form disulfide bonds, which can help
stabilize tertiary or the quaternary structure of
proteins, or serve as redox reaction.
Summary of some (by no means all) Functional Group Properties:
Aliphatics:
• Valine, Leucine, isoleucine, methionine, alanine.
• Provide hydrophobicity, structure. Important in folding.
Alcohols:
• Serine, threonine, tyrosine.
• Can be phosphorylated – critical in signaling
• Can be glycosylated, important for many functionals and structural aspects of
proteins.
• Important in many enzymatic reactions.
Aromatics:
• Tyrosine, phenylalanine, tryptophan (histidine also has aromatic ring properties).
• Structural, hydrophobic
• Often found in binding sites, stabilize other aromatic ring systems
Carboxylic acids:
• Glutamate and Aspartate
• Imparts charge

Bases:
• Lysine, Arginine, Histidine
• Impart charge
• Participate in enzymatic reactions.
Sulfhydryl:
• Cysteine
• Can form disulfide bonds – critical for tertiary structure and sometimes quarternary
structure.
• Similar to serine; also participates in enzymatic chemistry.

Structural Hierarchy
• Peptides and Proteins are covalent, linear chains of amino
acids. This defines their primary structure.
• Secondary, tertiary, and quaternary structure is acquired
through non-covalent interactions:
– Hydrogen bonding
– Hydrophobic interactions
– Ionic interactions
– Van der waals interactions
Amino acid chains – typically drawn Amino to Carboxy terminus.
Proteins have higher-order structures that are critical for function:
• Proteins could be chemically be described or defined through the primary sequence
alone. The primary structure is simply the sequence of amino acids and any
modifications made to them.
• Secondary structure is the first basic folding patterns that help proteins acquire
distinct structure. The three most important types of secondary structures are:
• Alpha-helices
• Beta sheets
• Turns/bends
• Tertiary structure is the assembly of secondary structures into subunit or into an
entire protein. This is a typically a discrete functional unit of a protein, and may be
the entire protein in some cases (eg cytochrome c, or myoglobin). Domains or folds
are patterns of tertiary folding that often recur in other proteins and form functional
structures within a protein or subunit. Again, some small proteins, like myoglobin or
cytochrome c have a single domain that constitutes the entire protein.
• Quaternary structure is the assembly of several peptides into a functional protein.
Usually each peptide is folded through secondary and tertiary (domains) prior to
assembly into a quaternary structure. A good example of a protein with a quaternary
structure is hemoglobin.

The Peptide Bond
• Peptides are linear chains of amino acids.
• The peptide, amide bond has double-bond character, and does not
rotate: This is a key aspect of higher order structure.
• There are two angles of rotation associated with each Amino
acid: N-Cα and Cα-CO. These are restricted by the R groups.
Peptide bonds between the Carboxyl group of one amino acid and the amine group of
the other. This forms an amide bond.
Amide bonds are relatively stable and require somewhat vigorous conditions to break
them chemically: boiling in 1 N acid (HCL) is typical for breaking all the peptide bonds in
a protein.
Amide bonds are planar and this is a key characteristic of protein structure. The
planarity is of 6 atoms:
On the C=O side:
The Carbonyl oxygen and carbon.
The alpha-carbon
On the NH side:
The N and the H
The alpha carbon on nitrogen side amino acid

Biosynthesis
• Central Dogma of Molecular
Biology
Review from Introduction to Medical Biochemistry
• Flow of information: DNA to mRNA to Protein
• This requires internal machinery of the cell to execute
• The various processes are compartmentalized (nucleus, cytosol, ER, etc)
• Each of these steps involves multiple layers of regulation that determines the extent
and location of protein expression.

The Genetic Code
• In the genetic code, each amino
acid is coded for by three
mRNA bases arranged in a
specific sequence.
• The first base in a codon is
found along the left side of the
chart.
• The second base is at the top of
the chart.
• The third base in the codon is
found along the right side of the
chart.
• There is one start codon (AUG)
and three stop codons (UAA,
UAG, UGA).
The genetic code.
You must be able to recognize the following:
• Stop codons
• Start codons
You must also be able to know how to use this chart.

InInInIn vivovivovivovivo peptide bond formation and growth of the
polypeptide chain
• The peptidyl
transferase is a
ribozyme.
• A ribozyme
is a RNA
molecule that
catalyzes a
chemical
reaction.
The polypeptide grows from the amino terminus to the carboxyl terminus.
• The amino acids in the ribosome are attached to their respective tRNAs by an ester
bond (R’CO - O - R) between the carboxyl terminus and either the 2’ or 3’ OH
groups of the ribose sugar of an adenosine.
• During formation of a peptide bond, the ester bond in the (P)eptidyl site is
cleaved, and peptidyl transferase catalyzes a condensation reaction between its
carboxyl terminus and the amino terminus of the amino acid in the (A)mino site.
• This transfers the P-site amino acid to the A-site amino acid.
• The polypeptide thus "grows" from the amino terminus to the
• carboxyl terminus.

Secondary Structure
• There are three different kinds
of secondary structures that
the amino acids can form.
• Beta sheet
• Alpha helix
• Random coil (turns)
Secondary Structure arises principally from wanting to satisfy Hydrogens Bonds on the
main chain carboxyl groups and amide groups.
Hydrogen bonds add + or - ~1 kcal/mol per bond to the stability of the secondary
structure element.
This is not a lot.
But, if a hydrogen bond is not formed (ie – no interaction with a main chain carbonyl or
amide), then there is a cost in stability of ~ +7 kcal/mol, unless it can interact with water
instead. So, failing to satisfy H-bonding is destabilizing. So, the protein will optimize H-
bonding in a manner consistent with the secondary structures to the extent possible.
The side chains, R-groups, determine what kind of secondary structure is favored.
Some proteins have all these secondary structures, while other proteins have
predominately one kind of secondary structure.

Secondary Structure – αααα-helices
α-Helices are right-hand helices stabilized by H-bonds running roughly parallel to the
helix axis
Certain amino acids preferentially form α-helices
The R-groups, side chains, all point out from the helix.
α-helices satisfy all backbone (main chain) Hydrogen bonds.
The α-helix is a rod-like structure with the peptide chain tightly coiled and the side
chains of amino acid residues extending outward from the axis of the spiral.
Each carbonyl group is hydrogen-bonded to the amide-hydrogen of a peptide
bond that is four residues away along the same chain.
There are 3.6 amino acids per turn.
The helix winds as a right-handed screw.

Secondary Structure – αααα-helices
• Examples
– Hemoglobin
– Rhodopsin
Hemoglobin
Two alpha-helical proteins, Hemoglobin and rhodopsin.
Rhodopsin is a transmembrane protein, whereas hemoglobin is completely soluble.

Secondary Structure – ββββ-sheets
• β-sheets form flat side-
by-side planes of amino
acids.
• The R-groups point up
and down (alternating)
from the plane of the
sheet.
• All main chain
hydrogen bonds are
satisfied, except edges
The β-pleated sheet is an extended structure as opposed to the coiled α-helix.
• Extended implies that Hydrogen bond interactions can occur among residues
distant in the primary sequence.
• In contrast, alpha-helices require hydrogen bonding to residues nearby in the
sequence (4 amino acids distant).
Beta-sheets:
• It is pleated because the alpha-carbon-carbon bonds are tetrahedral and cannot
exist in a planar configuration. This causes the structure to appear “pleated”
viewed from the side.
• If the polypeptide chains runs in the same direction, it forms a parallel β-sheet
• If the polypeptide chains runs in opposite direction, they form an antiparallel
structure.
• Parallel and anti-parallel beta-sheets have somewhat different hydrogen bonding
patterns.

Secondary Structure – ββββ-sheets
• Examples of β-
sheet proteins
– Porin
– IgG
• Also shows S-S
bonds
Beta-barrel Porin – A transmembrane protein
IgG structure – all beta sheet
Examples of beta-sheet proteins –
Beta barrels. This is an example of a porin – a transmembrane protein with a large pore.
Other beta-barrels exist in soluble proteins.
The IgG fold is a fold seen in many proteins but was first seen in crystal structures of
immunoglobulins.
Note that IgG is held together with disulfide bonds into its quaternary structure.
Note: Beta pleated sheet structures can be extensive and do not have to interact with
nearby residues. This differentiates them from alpha-helices where hydrogen bonding is
obligatorily to residues near in the sequence.

Tertiary Structure – 4 types of bonds
1. Hydrogen bonds
2. Hydrophobic interactions
3. Ionic bonds (rare)
4. Disulfide bridges
a. Only the disulfide bond is a
covalent bond.
b. Denaturing agents such as heat
will disrupt all these bonds
except the disulfide bond.
c. Disulfide bonds can be broken
by some reducing agents

Quaternary Structure: Hemoglobin versus myoglobin
Pedersen 2018 September 6 25Figures from http://slideplayer.com/slide/4462886/
Myoglobin: Monomeric protein
This is a single poly-peptide protein.
It has a single domain (globin fold)
It has primary, secondary and tertiary structure.
It does not have quaternary structure.
Hemoglobin: A tetrameric protein
This is made from 4 poly-peptide chains; 2 α, 2 β.
Each chain has a single domain (globin fold).
It has primary, secondary and tertiary structure.
It has quaternary structure
because 4 polypeptides are assembled into a
functional whole protein.
Quaternary Stucture: fully functional proteins assembled from individual subunits or
domains.
• Example:
• Hemoglobin (Hb) is the carrier of oxygen in the blood. It is assembled from four
subunits, two alpha and two beta. It has quaternary structure.
• Myoglobin (Mb) is the molecule that stores oxygen in tissues, such as muscle. It is a
single domain fold and a single peptide sequence. It has primary, secondary, and
tertiary structure, but not quaternary structure.
• Hemoglobin is composed of four proteins, 2 α-globin subunits and 2 β-globin
subunits, which affect one another to synergize the release or the binding of oxygen.
Because it is assembled from 4 subunits to form a functional whole, it has
quaternary structure.
• Myoglobin is similar in structure to the α-globin and β-globin protein momomers. It is
much smaller that hemoglobin, has a single peptide, and a single domain. Myoglobin
binds and releases oxygen in a non-cooperative fashion. It contains secondary
(alpha-helices) and tertiary (globin fold) structure, but no quaternary structure.
• These molecules are examples of proteins composed of α-helices only, and contain
no β-sheets.

Assembly of protein monomers into a quaternary structure provides added functionality
to proteins. In this example, the assembly of hemoglobin monomers into a tetramer
permits cooperativity between the binding sites. This cannot occur in a monomeric
protein such as myoglobin.

Henderson-Hasselbalch Equation
Acidic residues: pKa
Asp 3.9
Glu 4.3
Basic Residues:
Lys 10.5
Arg 12.5
His 6.0
Cysteine
Cys 8.3
-COOH -COO-
Fraction
AH
pH:
Acid Base Chemistry:
The amino and carboxylic acid pKa values on each amino acids are only relevant for
the two residues at the ends. The rest are in amide bonds and not tritratable.
For most large proteins, the end amino acids are a minor contribution to the over-all
charge properties.
pKa of a terminal Amino group is about 9 to 10 for individual amino acids.
pKa of a terminal Carboxylate group is about 2.0, for individual amino acids.
More important are the side chain pKa values – the determine the overall charge and
solubility of a protein.
The most important ones are listed above. Also relevant is the tyrosine side chain:
Tyrosine pKa ~ 10.
The graph above shows the fraction of AH still present as a function of pH. It is a
titration curve, as you add increasing amounts of base.
The Henderson–Hasselbalch equation describes the relation of pH to the acid (AH)
and conjugate base forms (A-) of a titratable group, in biological systems.

(using pKa, the negative log of the acid dissociation constant)
The equation can be used to do the following:
Estimate the pH of a buffer solution
Find the equilibrium pH in acid-base reactions
Calculate the isoelectric point of proteins
pKa is the pH where the acid and conjugate base forms of a compound are equal in
concentration.
Buffering Capacity
The pKa is the best buffering point of a pH buffer.
The buffering capacity of a solution is the change in pH for the amount of acid (or base)
added. The less change in pH, the better the buffering. Imagine a titration experiment
where you have a solution of a buffering compound (eg Histidine or phosphate), and
you then slowly add acid (or base). Basically, at the pKa, the titratable compound soaks
up the added acid or base, and the pH changes little. Once the pH moves away from
the pKa, the compound is fully titrated to its acid or base form, and then any further
added acid or base, will give a large change in H+ concentration; it no longer has
buffering capacity.
Isoelectric Point
The isoelectric point, pI, is the pH value where the molecule is net neutral (net charge is
zero). The pI value is less important for individual amino acids than the protein as a
whole. The pH that proteins are net neutral at often makes them less soluble, and they
may precipitate out of solution.
Most proteins (but by no means all), are more acidic, meaning that they have a pI
of less than 7; typical values are 5-6. However, the pI values can range up to basic
values near the pKa of lysine. This just depends on the number of acidic and basic
residues in the protein.
The pI can be calculated precisely from the pKa’s of the all the individual side
chains using the HH equation for all of them. This requires a computer program and can
be done on the web. A quick, and relatively good estimation can be made from the
primary sequence as follows:
• Guess at a pI by looking at the number of basic and acidic residues.
• Count the charges on the protein at the pH you guessed for the pI (if you guess pI =
4.5, assume pH is 4.5).
• If you are close to the pKa of a residue, assign a partial charge. This doesn’t
have to be precise – just round to nearest quarter charge.
• If you have negative net charge, move the guess for the pI lower.
• If you have a negative positive charge, move the guess for the pI higher.
• Repeat steps 2 to 4 until the net charge is near zero.
• Find the two pKa’s from all the residues that most closely bracket your current
guess for the pI.
• Average those two values.

• E.g. if your pI guess is 5.2, you find you have a Glu in your sequence and a
His, and these are closes to your pI, then average those pKa’s: (4.3 + 6.0)/2
= 5.15. This would be your best guess for the pI.
Key Points:
You have to know how to use the Henderson-Hasselbalch equation. Do some practice
problems.
Know what a pI is
Know what a pKa is, how it relates to buffering capacity and a titration curve.
Know what the charge would be on the important side chains at normal and acidic and
basic pH values.
Know what buffering capacity means and where a compound buffers best.

Asp 3.9
Glu 4.3
Basic Residues:
Lys 10.5
Arg 12.5
His 6.0
Cysteine
Cys 8.3
-NH3
+ -NH2
Fraction
AH
pH:

Asp 3.9
Glu 4.3
Basic Residues:
Lys 10.5
Arg 12.5
His 6.0
Cysteine
Cys 8.3
-NH3
+ -NH2
Fraction
AH
pH:
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
pK = 6.0

Asp 3.9
Glu 4.3
Basic Residues:
Lys 10.5
Arg 12.5
His 6.0
Cysteine
Cys 8.3
-SH -S-
Fraction
AH
pH:

Asp 3.9
Glu 4.3
Basic Residues:
Lys 10.5
Arg 12.5
His 6.0
Cysteine
Cys 8.3
-COOH -COO-
-SH -S-
-NH3
+ -NH2
Fraction
AH
pH:

Net Protein Charge
• The overall charge of a polypeptide/protein is the sum total of all the + and -
charges on the side chains of the amino acids constituting the protein.
• For instance, the following polypeptide would have the following charges, at pH 7.4,
on the side chains of the amino acids in the polypeptide:
+1 0 -1 0 +1 +1 0 -1 -1 0 0 -1 -1
• +NH3-Val-Glu-Pro-Arg-Lys-Ile-Asp-Glu-Gln-Thr-Glu-C00-
• Note that the N-terminus is always a +1 at pH 7.4 and the C-terminus is always a -1
at pH
7.4, unless they are modified.
• The net charge on this peptide is -2 = 5(-1 charges) + 3(+1 charges).Pedersen 2018 September 6 31
How to count charges:
• Determine the pH you are working at.
• For acidic residues, if pH << pKa, count zero, if pH >> pKa, count -1.
• For basic residues, , if pH << pKa, count +1, if pH >> pKa, count zero.
• If the pH is near pKa, count 0.5 charge (+0.5 for basic, -0.5 for acidic residues). This
applies especially to Histidine, where it is typically counted as +0.5 at neutral pH; but
can also occur with cysteine (pKa 8.3, count as negative charge).
Note: These are approximations, even if you are careful. This is because the protein
environment can change the pKa of the side chains. So, in the context of a large
protein, the given side chain pKa’s are just good estimates. The environment can alter
them. This happens a lot in active sites.
For example, Histidine is often found in the active sites of proteases and
esterases. The local enclosed environment of the site makes the pKa closer to neutrality
where it can effectively work as an acid or base.

Post-translational modification – an example
• There are many modifications
• Phosphorylation is one example
• Often serves a regulatory roles
• Other modifications can serve other roles
Once a peptide/protein has been created, the amino acids can be further modified, e.g.
Phosphorylation: -PO4
=
Acetylation: -COCH3
Methylation: -CH3
Hydroxylation: -OH
Glycosylation: various sugar residues, sometimes many sugars
Cyclic phosphorylation and dephosphorylation is a common cellular mechanism for regulating
protein activity.
In this example, a target protein R is inactive when phosphorylated and active when
dephosphorylated.
In some proteins, the opposite is the case.

An enzyme: Cu,Zn-superoxide dismutase
• Superoxide dismutase has a dimeric structure,
with a monomer molecular mass of 16,000 Da.
Cu and Zn are cofactors.
• Each subunit consists of eight antiparallel β-
sheets called a β-barrel structure.
• Superoxide dismutase catalyzes the
dismutation of the superoxide (O2
.-) radical into
either molecular oxygen, O2 or to hydrogen
peroxide, H2O2.
Many Proteins are enzymes.
Enzymes are biological catalysts.
In cells and organisms most reactions are catalysed by enzymes which are regenerated during
the course of a reaction.
These biological catalysts are physiologically important because they speed up the rates of
reactions.
Enzymes are used in the diagnosis of pathology.
The measurement of the serum levels of numerous enzymes is used diagnostically.
This is because the presence of these enzymes in the serum indicates that tissue or cellular
damage has occurred resulting in the release of intracellular component into the blood.
An example enzyme: Superoxide dismutase (SOD).
Enzymes often have cofactors.
Superoxide dismutase (SOD) has several metal cofactors that help catalyze the reaction.
SOD is a mulimeric enzyme. Functionally it is dimeric with two catalytic sites. It is symmetric.
The x-ray crystal structure actually shows two dimers here.

Collagen
• Collagen
– Major Structural protein
– Unusual Triple Helical structure
– Has high % Gly and Pro
– Atypical 2nd and quaternary
structure
– Other structural proteins also rely
on structures atypical for globular
proteins.
Collagen:
• Collagens are the most abundant protein family in our body representing about
1/3 of the body proteins.
• Collagens are a major component of connective tissue such as cartilage,
tendons, the organic matrix of bones, and the cornea of the eye.
• Collagen contains 35% Gly and 21% Pro plus Hyp (hydroxyproline).
• The amino acid sequence in collagen is generally a repeating tripeptide unit, Gly-
Xaa-Pro or Gly-Xaa-Hyp, where Xaa can be any amino acid.
• The repeating amino acids adopt a left-handed helical structure with 3 aa per turn.
• Three of these helices wrap around one another with a right-handed twist to form
tropocollagen.
• Tropocollagen molecules self-assemble into collagen fibrils and are packed
together to form collagen fibers.
• Scurvy, osteogenesis imperfecta, and Ehlers-Danlos syndrome result defects
in collagen synthesis and/or crosslinking.
To mature collage, prolines on collagen are converted to hydroxyproline. This is
carried out by the enzyme prolyl hydroxylase.
Prolyl hydroxylase requires vitamin C, ascorbate, for it’s activity.
If you have a deficiency in vitamin C, you will be unable to resynthesize collagen,
and get the disease scurvy.

Summary
• Amino acids constitute proteins according to the genetic code.
• Amino acids contain an α-carbon that is covalently linked to an amino group, a carboxylic
acid group, and a distinctive R group, or side chain group. Only the L-amino acid
stereoisomers are used by the ribosome.
• Proteins are composed of 20 amino acids, designated by a 3-letter code or a 1-letter code.
• The Henderson–Hasselbalch equation can be used to determine the charge on side chains.
• The pKa is the pH at which a functional group will buffer the best.
• The overall charge of a polypeptide/protein is the sum total of all the + and – charges.
• The primary structure of a protein is its sequence of amino acids.
• The secondary structure of a protein typically defined through hydrogen bond interactions
and can take 3 forms: alpha helix, a beta-pleated sheet, and a random coil.
• The tertiary structure of a protein is held together by several kinds of interactions:
Hydrophobic interactions, van der waals interactions, ionic bonds, disulfide bridges.

Protein structureandfunction 2018_fall3 (1)

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