Transcript Section 5.3: Proteins - Clayton State University

Online Video
Amino Acids
Section 5.1, page 132
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
Classification of
Amino Acids
Section 5.1, page 134
How amino acids can be classified
according to their side chain’s
chemical properties.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
Peptide Bond
Formation and
Cleavage
Section 5.1, page 142
How is the peptide bond formed and
how can it be cleaved?
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
What is a disulfide
bond?
Section 5.1, page 144
What is a disulfide bond, and how are
they formed?
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
Amino Acids and
Protein Structure
Section 5.3, page 149
Structure and properties of amino
acids: backbone, side chain,
hydrophobic and hydrophilic
properties. Formation of a
polypeptide: primary, secondary,
tertiary, and quaternary structure.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
Four Levels of
Protein Structure
Section 5.3, page 149
The different levels of protein
structure – primary, secondary,
tertiary, and quaternary.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
Protein Folding
and Denaturation
Section 5.3, page 161
Which forces contribute to protein
structure and how are these forces
disrupted?
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
The Protein
Folding Problem
Section 5.3, page 163
For 50 years, the “protein folding
problem” has been a major mystery.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
Hemoglobin
Section 5.3, page 172
Hemoglobin and its role in the
circulatory system.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
Motor Proteins
Section 5.4, page 176
The two-legged molecules known as
motor proteins are what get the job of
living done in most of your cells.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Online Video
A Day in the Life
of a Motor Protein
Section 5.4, page 176
A motor protein has to transport its
package to the right destination in the
nerve cell, illustrating the relevance
and mechanisms of proper
intracellular transport in the nervous
system.
From McKee and McKee, Biochemistry, 6th Edition, © 2016 by Oxford University Press
Chapter 5
Amino Acids, Peptides, and Proteins
Overview




Section 5.1: Amino Acids
Section 5.2: Peptides
Section 5.3: Proteins
Section 5.4: Molecular
Machines
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Figure 5.1 Protein Diversity
Proteins are molecular
tools
They are a diverse and
complex group of
macromolecules
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Structures of Amino Acids

Proteins and polypeptides are biochemical compounds
consisting of amino acids



Chains of amino acids bonded together by peptide bonds
Amino acid polymers of 50 or less are peptides; polymers
greater than 50 are proteins or polypeptides
Proteins



Longer and more complex than polypeptides
Typically folded into a globular or fibrous form
Structure facilitates a biological function
Peptide linkages
R
R
H 3N
O
+
+
H3N
O
-
Amino acid
CH
NH
C
CH
O
R
O
R
C
CH
NH
Polypeptide
O
-
C
O
Protein
Amino Acids





Organic compounds with amino and carboxylate functional groups
Each AA has unique side chain (R) attached to alpha (α) carbon
Crystalline solids with high MP’s
Highly-soluble in water
Exist as dipolar, charged zwitterions (ionic form)
Seager SL, Slabaugh MR, Chemistry for Today: General, Organic and Biochemistry, 7 th Edition, 2011; Berg JM, Tymoczko JL, Stryer L, Biochemistry, 5th Edition, 2002
Section 5.1: Amino Acids
Figure 5.3 General
Structure of the aAmino Acids
19 have the same general
structure: central (a) carbon, an
amino group, carboxylate group,
hydrogen atom, and an R group
(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
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Figure 5.2 The
Standard Amino
Acids
Amino Acid Classes
Classified by their ability to interact with water
Nonpolar amino acids contain hydrocarbon groups
with no charge
https://www.youtube.com/watch?v=lxD4819UvaQ
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Figure 5.2 The Standard Amino Acids
Amino Acid Classes Continued
Polar amino acids have functional groups that can
easily interact with water through hydrogen bonding
Contain a hydroxyl group (serine, threonine, and
tyrosine) or amide group (asparagine)
https://www.youtube.com/watch?v=McKprZI0DXM
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Figure 5.2 The
Standard Amino
Acids
Amino Acid Classes Continued
Acidic amino acids have side chains with a carboxylate
group that ionizes at physiological pH
Basic amino acids bear a positive charge at physiological
pH
At physiological pH, lysine is its conjugate acid
(-NH3+), arginine is permanently protonated, and histidine
is a weak base, because it is only partly ionized
https://www.youtube.com/watch?v=OPAvXQsPCqQ
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Other Biological Roles of Amino Acids
1. Some amino acids or derivatives can act as
chemical messengers
Neurotransmitters (serotonin derived from
tryptophan) and hormones (thyroxine derived from
tyrosine)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
2. Act as precursors for other
molecules (nucleotides and
heme)
3. Metabolic intermediates
(arginine, ornithine, and
citrulline in the urea cycle)
Figure 5.5 Citruline
and Ornithine
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Figure 5.6 Modified
Amino Acid Residues
Found in Polypeptides
Modified Amino Acids in Proteins
AAs can be modified after protein synthesis
Serine, threonine, and tyrosine can be phosphorylated
g-Carboxyglutamate (prothtrombin), 4-hydroxyproline
(collagen), and 5-hydroxylysine (collagen)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Amino Acid Stereoisomers
Because the a-carbon (chiral carbon) is attached to
four different groups, they can exist as stereoisomers
Except glycine, which is the only nonchiral standard
amino acid
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Exist as either L- or Denantiomers
They cannot be
superimposed over one
another and rotate plane,
polarized light in opposite
directions (optical isomers)
Almost without exception,
biological organisms use only
the L enantiomer
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Titration of Amino Acids
Free amino acids contain ionizable groups
https://www.youtube.com/watch?v=ffKS1ev1HD0
The ionic form depends on the pH
When amino acids have no net charge due to
ionization of both groups, this is known as the
isoelectric point (pI) and can be calculated using:
pK1 + pK2
pI =
2
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.1: Amino Acids
Alanine is a simple amino
acid with two ionizable
groups
Alanine loses two protons in
a stepwise fashion upon
titration with NaOH
Isoelectric point is reached
with deprotonation of the
carboxyl group
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
pH vs. pKa
When pH is less than pKa, side chain is
protonated.
 When pH is greater than pKa, side
chain is deprotonated.
 As pH gets lower, concentration of H+
increases, more H+ is available to
protonate residues.
 At physiological pH (7.4), Asp is
deprotonated (pKa = 3.90) and Lys is
protonated (pKa = 10.79)

Section 5.1: Amino Acids
Amino acids with ionizable side
chains have more complex titration
curves
Figure 5.9 Titration of Two
Amino Acids: Glutamic Acid
Glutamic acid is a good example,
because it has a carboxyl side chain
group
Glutamic acid has a +1 charge at
low pH
Glutamic acid’s isoelectric point
as base is added and the acarboxyl group loses a proton
As more base is added, it loses
protons to a final net charge of -2
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Formation of Polypeptides

Polypeptides and proteins are created through formation
of peptide bonds between amino acids

Condensation or dehydration reaction; amide formation
Peptide linkages
R
+
H3N
CH
NH
C
CH
O
R
O
R
C
CH
NH
Polypeptide
http://en.wikipedia.org/wiki/File:AminoacidCondensation.svg
O
C
O
-
Section 5.1: Amino Acids
Linus Pauling was the first to
characterize the peptide bond as
rigid and flat
Found that C-N bonds between
two amino acids are shorter
than other C-N bonds
Gives them partial doublebond characteristics (they
are resonance hybrids)
Figure 5.11 The Peptide Bond
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Resonance Tautomers of a Peptide
Properties of the Peptide
Bond


Each peptide unit contains the Ca atom and the C'=O group of the
residue n as well as the NH group and the Ca atom of the residue
n + 1.
Each unit is a planar, rigid group with known bond distances and
bond angles. R1, R2, and R3 are the side chains attached to the
Ca atoms that link the peptide units in the polypeptide chain.
Section 5.1: Amino Acids
Because of the rigidity, onethird of the bonds in a
polypeptide backbone cannot
rotate freely
Limits the number of
conformational possibilities
Figure 5.11 The Peptide Bond
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Disulfide Bridge



Disulfide bonds form between
side chains of two cysteine
residues.
Two SH groups from cysteine
residues, which may be in
different parts of the AA
sequence but adjacent in the
3D structure, can be oxidized
to form one S-S (disulfide)
group.
Usually occurs in
extracellular proteins.
2 -CH2SH + 1/2 O2  -CH2-S-S-CH2 + H2O
Section 5.1: Amino Acids
Schiff Base Formation: imine products of primary
amine groups interacting with carbonyl groups
Most important examples are amino acid biosynthesis
reactions
Schiff bases, referred to as aldimines, are
intermediates formed by the reaction of an amino
group with an aldehyde group
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.2: Peptides
Less structurally complex than larger proteins,
peptides still have biologically important functions
Vasopressin is an antidiuretic hormone that
regulates water balance, appetite, and body
temperature
Oxytocin is a peptide that aids in uterine
contraction and lactation
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Of all molecules in a living organism, proteins
perform the most diverse functions:
Catalysis (enzymes)
Structure (cell and organismal)
Movement (amoeboid movement)
Defense (antibodies)
Regulation (insulin is a peptide hormone)
Transport (membrane transporters)
Storage (ovalbumin in bird eggs)
Stress Response (heat shock proteins)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Protein Functions

Catalytic Function:


Enzymes are proteins that catalyze biological functions
Structural function:

Most human structural materials (excluding bone) are comprised of proteins
 Collagen (bundled helices)
 25-35% of total protein in body
 Tendons
 ligaments
 Skin
 Cornea
 Cartilage
 Bone
 blood vessels
 gut
 Keratin (bundled helices)
 Chief constituent of hair, skin, fingernails
http://www.imb-jena.de/~rake/Bioinformatics_WEB/proteins_classification.html
Protein Functions

Storage Function:

Storage of small molecules or ions
 Ovalbumin
 Main protein in egg whites
 Can be broken down into amino acids for use by developing embryos
 Ferritin
 Globular complex of 24 protein subunits
 Buffers iron concentration in cells
Ovalbumin (chicken egg white)
http://www.stagleys.demon.co.uk/explorers/genesandproteins/page6.html; http://ferritin.blogspot.com/
ferritin
Protein Functions

Protective Function:
 Protection against external foreign
substances

Immunoglobulin
Antibodies
Very large proteins
Combine with, and destroy viruses, bacteria
blood clotting/Coagulation

–
• thrombin
– Protease responsible for platelet aggregation
and formation of fibrin
Harris, L. J., Larson, S. B., Hasel, K. W., Day, J., Greenwood, A., McPherson, A. Nature 1992, 360, 369-372; http://courses.washington.edu/conj/immune/antibody.htm;
http://www.colorado.edu/intphys/Class/IPHY3430-200/014blood.htm
Protein Functions

Regulatory Function:
 Protein hormones

Insulin


Protein hormone that directs cells in the liver,
muscle, and fat to take up glucose from the blood
and store it as glycogen
Forms hexamer bound together by Zn
Insulin
http://en.wikipedia.org/wiki/File:InsulinHexamer.jpg; Seager SL, Slabaugh MR, Chemistry for Today: General, Organic and Biochemistry, 7 th Edition, 2011
Protein Functions

Nerve impulse transmission:
 Rhodopsin

Protein found in rods cells of eye retina

Converts light events into nerve impulses sent to
the brain
http://cherfan2010biology12assessment.wikispaces.com/The+Retina
Protein Functions

Movement function:
 Proteins involved in muscle contraction


Myosin
Actin
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/structural-proteins/actin.html
Protein Functions

Transport function:
 Transport ions or molecules throughout the body



Serum albumin: Transports fatty acids between fat and other tissues
Hemoglobin: Transports O2 from lungs to other tissues (e.g., muscles)
Transferrin: Transports iron in blood plasma
Serum albumin
hemoglobin
transferrin
http://en.wikipedia.org/ ; http://www.pdb.org/pdb/101/motm.do?momID=37
Section 5.3: Proteins
Due to recent research, numerous multifunction
proteins have been identified
Proteins are categorized into families based on
sequence and three-dimensional shape
Superfamilies are more distantly related proteins
(e.g., hemoglobin and myoglobin to neuroglobin)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Protein Classifications
Based on structural shape

Fibrous Proteins

Comprised of long stringlike molecules that can wrap around each other to form fibers
 Usually insoluble in water
 Major components of connective tissues (e.g., collagen, keratin)

Globular proteins

Spherical
 Usually water soluble
 May be moved through the body (e.g., hemoglobin, transferrin)
Based on composition
• Simple Proteins
– Contain only amino acid residues
• Conjugated Proteins
– Contain amino acid residues and other organic or inorganic components (i.e., prosthetic
groups)
• Lipoproteins
• Glycoproteins
• metalloproteins
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/structural-proteins/actin.html
Section 5.3: Proteins
Protein Structure
Proteins are
extraordinarily complex;
therefore, simpler images
highlighting specific
features are useful
Space-filling and ribbon
models
Figure 5.14 The Enzyme
Adenylate Kinase
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Proteins have different levels of structure




Primary (1°): Sequence of amino
acids
 Determines 3D structure
Secondary (2°): H-bonding
interactions between AA residues
begin to produce regular,
identifiable structures
 Alpha (α) helices
 Beta (β) strands
 Random coil
Tertiary (3°): Overall structure of
single protein in 3 dimensions
Quaternary (4°): Assemblies of
multiple polypeptides and/or
proteins
http://protein-pdb.com/2011/10/04/primary-protein-structure/
Section 5.3: Proteins
Figure 5.15 Segments of b-chain in HbA and HbS
Primary Structure is the specific amino acid
sequence of a protein
Homologous proteins share a similar sequence and
arose from the same ancestor gene
When comparing amino acid sequences of a protein
between species, those that are identical are invariant
and presumed to be essential for function
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Primary Structure, Evolution, and Molecular
Diseases
Due to evolutionary processes over time, the amino
acid sequence of a protein can change due to alterations
in DNA sequences called mutations
Many mutations lead to no change in protein function
Some sequence positions are less stringent
(variable) because they perform nonspecific
functions
Some changes are said to be conservative, because it is
a change to a chemically similar amino acid
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.15 Segments of b-chain in HbA and HbS
Primary Structure, Evolution, and Molecular
Diseases Continued
Mutations can be deleterious, leading to molecular
diseases
Sickle cell anemia is caused by a substitution of valine
for a glutamic acid in b-globin subunit of hemoglobin
Valine is hydrophobic, unlike the charged glutamic
acid
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Primary Structure, Evolution,
and Molecular Diseases
Continued
The substitution for
hydrophobic valine HbS:
molecules aggregate to form
sickle-shaped cells
These cells have low oxygenbinding capacity and are
susceptible to hemolysis
Figure 5.16 Sickle Cell Hemoglobin
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Protein Secondary Structure
Seager SL, Slabaugh MR, Chemistry for Today: General, Organic and Biochemistry, 7 th Edition, 2011
Section 5.3: Proteins
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
Figure 5.17 The a-Helix
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Proteins 2° Structure: The α-helix



Backbone N-H groups form H-bonds with C=O group four residues away in
sequence
AA’s in an α helix arranged in a right-handed helix
Each amino acid residue is rotated 100° relative to previous residue in helix
 Helix has 3.6 residues per turn
Glycine
and proline
do not
foster
a-helical
formation
http://simplygeology.wordpress.com/tag/s-waves/
Proteins 2° Structure: The β-sheet




Beta (β) sheets formed by H-bond connected strands
β strands are elongated helices without helical H-bonds
β Sheets may be parallel or antiparallel
Antiparallel sheets are more stable
http://www.chembio.uoguelph.ca/educmat/phy456/456lec01.htm
Proteins 2° Structure: Random Coils and Loops

Proteins typically contain regions lacking either sheet or helical
structures. These regions may be classified as:

Random Coils
 Loops

Loops may perform important structural and functional roles,
including:
Connecting β strands form antiparallel sheets
 Increasing flexibility (hinge motion)
 Binding metal ions or other biomolecules to alter protein function

http://www.chembio.uoguelph.ca/educmat/phy456/456lec01.htm
Section 5.3: Proteins
Figure 5.19 Selected Supersecondary Structures
Many proteins form supersecondary structures
(motifs) with patterns of a-helix and b-sheet structures
(a) bab unit
(b) b-meander
(c) aa unit
(d) b-barrel
(e) Greek key
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Tertiary Structure refers to unique threedimensional structures formed by globular proteins
Also prosthetic groups
Protein folding is the process by which a nascent
molecule acquires a highly organized structure
Information for folding is contained within the
amino acid sequence
Interactions of the side chains are stabilized by
electrostatic forces
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Proteins 3° Structure


Protein function determined by 3D shape
Tertiary structure results from residue interactions:





H-bonding
Disulfide Bridges
Salt Bridges
Hydrophobic Interactions
Hydration
Seager SL, Slabaugh MR, Chemistry for Today: General, Organic and Biochemistry, 7 th Edition, 2011
Proteins 3° Structure

Polar and charged residues tend to be on surface of protein, exposed
to water, while hydrophobic residues tend to be buried
Seager SL, Slabaugh MR, Chemistry for Today: General, Organic and Biochemistry, 7 th Edition, 2011
Section 5.3: Proteins
3. Large globular proteins (200+
amino acids) often contain
several domains
Domains are structurally
independent segments that
have specific functions
Core structural element of a
domain is called a fold
Figure 5.20 Selected
Domains Found in Large
Numbers of Proteins
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Conformational Changes of Calmodulin
Ca Ca
Ca Ca
Peptide
3bya
3cln
1cfc
EF-I
helix
l
o
o
p
helix
Ikura et al., Science, 1992. 256(5057): p. 632-8.; Yuan et al,. J Biol Chem, 1999. 274(13): p. 8411-20.; Zhou et al., Cell Calcium,
2009. 46(1): p. 1-17. Goldstein and Ar, Life Sci, 1983. 33(10): p. 1001-6. Chao et al., Arch Toxicol, 1995. 69(3): p. 197-203.
Section 5.3: Proteins
Figure 5.20 Selected Domains Found in Large Numbers
of Proteins
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.20 Selected Domains Found in Large Numbers of
Proteins
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.21 Fibronectin Structure
4. A number of proteins called mosaic or modular
proteins consist of repeated domains
Fibronectin has three repeated domains (F1, F2,
and F3)
Domain modules are coded for by genetic
sequences created by gene duplications
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.24 Structure of
Immunoglobulin G
Quaternary structure: a protein that is composed of
several polypeptide chains (subunits)
Multisubunit proteins may be composed, at least in
part, of identical subunits and are referred to as
oligomers (composed of protomers)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Proteins 4° Structure

Functional proteins may
contain two or more
polypeptide chains held
together by the same forces
that control 3° structure:

H-bonding
 Disulfide Bridges
 Salt Bridges
 Hydrophobic Interactions


Seager SL, Slabaugh MR, Chemistry for Today: General, Organic and Biochemistry, 7 th Edition, 2011
Each chain is a subunit of
structure
Each subunit has its own 1°,
2° and 3° structure
Section 5.3: Proteins
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
Figure 5.24 Structure of
Immunoglobulin G
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Interactions between subunits are often affected by
ligand binding
An example of this is allostery, which controls protein
function by ligand binding
Can change protein conformation and function
(allosteric transition)
Ligands triggering these transitions are effectors
and modulators
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Allosteric Regulation Models

(a) Cooperativity: a cartoon representation of the Monod-Wyman-Changeux
(MWC) model of allosteric transitions. A symmetric, multimeric protein can
exist in one of two different conformational states—the active and inactive
conformations. Each subunit has a binding site for an allosteric effector as
well as an active site or binding site.
Allosteric regulation and catalysis emerge via a common route, Nature Chemical Biology, 2008
Allosteric Regulation Models

(b) A monomeric, allosterically inhibited protein. The binding of an allosteric
inhibitor alters the active site or binding site geometry in an unfavorable
way, thereby decreasing affinity or catalytic efficiency.
Allosteric regulation and catalysis emerge via a common route, Nature Chemical Biology, 2008
Allosteric Regulation Models

(c) A monomeric, allosterically activated protein. The binding of an allosteric
activator results in increased affinity or activity in the second site.
Allosteric regulation and catalysis emerge via a common route, Nature Chemical Biology, 2008
Allosteric Regulation Models

(d) The binding of an allosteric effector might introduce a new binding site to
a protein. Binding of a ligand to this new binding site could lead to changes
in active site geometry, providing an indirect mechanism of allosteric
control. This type of effect is of great interest in the design of allosteric
drugs and can be considered a subset of the example shown in c.
Allosteric regulation and catalysis emerge via a common route, Nature Chemical Biology, 2008
Allosteric Regulation Models

(e) The fusion of an enzyme (maroon) to a protein under allosteric control.
This type of construct can act as an allosteric switch because the activity of
the enzyme is indirectly under allosteric control via the bound protein with
an allosteric site. Such constructs are both present in nature and the target
of protein engineering studies.
Allosteric regulation and catalysis emerge via a common route, Nature Chemical Biology, 2008
Section 5.3: Proteins
Figure 5.26 Disordered
Protein Binding
Unstructured proteins: Some proteins are partially
or completely unstructured
Unstructured proteins referred to as intrinsically
unstructured proteins (IUPs) or natively unfolded
proteins
Often these proteins are involved in searching out
binding partners (i.e., KID domain of CREB)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.27 The Anfinsen
Experiment
Loss of Protein Structure: Because of small
differences between the free energy of folded and
unfolded proteins, they are susceptible to changes in
environmental factors
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.27 The Anfinsen
Experiment
Disruption of protein structure is denaturation
(reverse is renaturation)
Denaturation does not disrupt primary protein
structure
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Denaturing Conditions:
1. Strong acid or base
2. Organic solvents
3. Detergents
4. Reducing agents
5. Salt concentration
6. Heavy metal ions
7. Temperature
8. Mechanical stress
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
The Folding Problem
The direct relationship between a protein’s primary
sequence and its final three-dimensional conformation
is among the most important assumptions in
biochemistry
http://youtu.be/zm-3kovWpNQ
Painstaking work has been done to be able to predict
structure by understanding the physical and chemical
properties of amino acids
X-ray crystallography, NMR spectroscopy, and sitedirected mutagenesis
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Important advances have
been made by biochemists in
protein-folding research
This research led to the
understanding that it is
not a single pathway
A funnel shape best
describes how an unfolded
protein negotiates its way to a
low-energy, folded state
Numerous routes and
intermediates
Figure 5.28 The Energy Landscape
for Protein Folding
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Small polypeptides (<100
amino acids) often form
with no intermediates
Larger polypeptides often
require several
intermediates (molten
globules)
Many proteins use
molecular chaperones to
help with folding and
targeting
Figure 5.29 Protein Folding
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Molecular chaperones assist
protein folding by protecting
them from inappropriate proteinprotein interactions:
Four groups: RibosomeAssociated Chaperones, Hsp70s,
Hsp90s, and chaperonins.
Figure 5.30 Space-Filling
Model of the E. Coli
Chaperonin, called the
GroES-GroEL Complex
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Hsp70s are a family of
chaperones that bind and
stabilize proteins during
the early stages of folding
Chaperonins increase
speed and efficiency of the
folding process
Figure 5.31 The Molecular Chaperones
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Both use ATP hydrolysis
Both are also involved in
refolding proteins
If refolding is not possible,
molecular chaperones
promote protein degradation
Figure 5.31 The Molecular
Chaperones
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Fibrous Proteins
Typically contain high
proportions of a-helices
and b-pleated sheets
Often have structural
rather than dynamic
roles and are water
insoluble
Keratin (a-helices) and
silk fibroin (b-sheets)
Figure 5.32 a-Keratin
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Collagen: part of the connective
tissue matrix synthesized by
connective tissue and secreted
into the extracellular space
20 major families with diverse
functions
Impart special properties to
structures (e.g., bone and skin)
Glycine and proline are
common amino acids
Figure 5.34 Collagen Fibrils
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Globular Proteins
Biological functions often include
precise binding of ligands
Myoglobin and hemoglobin
Both have a specialized heme
prosthetic group used for
reversible oxygen binding
Figure 5.35 Heme
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Myoglobin: found in high
concentrations in cardiac
and skeletal muscle
The protein component
of myoglobin, globin, is a
single protein with eight
a-helices
Encloses a heme [Fe2+]
that has a high affinity
for O2
Figure 5.36 Myoglobin
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Hemoglobin is a roughly spherical
protein found in red blood cells
Figure 5.37 The OxygenBinding Site of Heme
Created by a Folded
Globin Chain
Primary function is to transport
oxygen from the lungs to tissues
HbA molecule is composed of 2
a-chains and 2 b-chains (a2b2)
2% of hemoglobin contains dchains instead of b-chains (HbA2)
Embryonic and fetal hemoglobin
have e- and g-chains that have a
higher affinity for O2
https://www.youtube.com/watch?v=L
WtXthfG9_M
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.39 Hemoglobin
Comparison of myoglobin and hemoglobin identified
several invariant residues, most having to do with
oxygen binding
Four chains of hemoglobin arranged as two identical
ab dimers
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.40 The Hemoglobin
Allosteric Transition
Oxygenation of hemoglobin causes the dimers to slide
by each other and rotate 15º
Deoxygenated hemoglobin is considered the T (taut)
state and oxygenated is the R (relaxed) state
Hemoglobin alternates between the two stable states
T and R
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.38 Dissociation
Curves Measure the
Affinity of Hemoglobin and
Myoglobin for Oxygen
Hemoglobin shows a sigmoidal oxygen dissociation
curve due to cooperative binding
Binding of first O2 changes hemoglobin’s
conformation making binding of additional O2 easier
Myoglobin dissociation curve is a hyperbolic simpler
binding pattern
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Binding of ligands other than oxygen affects
hemoglobin’s oxygen-binding properties
pH decrease enhances oxygen release from
hemoglobin (Bohr effect)
The waste product CO2 (which is converted to
carbonic acid) also enhances oxygen release by
increasing H+ concentration
Binding of H+ to several ionizable groups on
hemoglobin shifts it to its T state (deoxygenated)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.3: Proteins
Figure 5.41 The Effect of 2,3Bisphosphoglycerate (BPG) on
the Affinity Between Oxygen
and Hemoglobin
2,3-Bisphosphoglycerate (BPG) is also an important
regulator of hemoglobin function
Red blood cells have a high concentration of BPG,
which lowers hemoglobin’s affinity for O2
In the lungs, these processes reverse
RBCs bind oxygen better in lungs, release better
when not in lungs
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Section 5.4: Molecular Machines
Molecular Machines
Purposeful movement is a hallmark of living things
This behavior takes a myriad of forms
Biological machines are responsible for these behaviors
Usually ATP or GTP driven
Motor proteins fall into the following categories:
• Classical motors (myosins, dyneins, and
kinesin)
• Timing devices (EF-Tu in translation)
• Microprocessing switching devices (G proteins)
• Assembly and disassembly factors (cytoskeleton
assembly and disassembly)
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Biochemistry in the Lab
 Protein Technology
Many techniques available to study proteins
Purification: Protein analysis starts with isolation
and purification
First step may be salting out of a protein fraction
Figure 5E Dialysis
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Biochemistry in the Lab
Chromatography can
separate protein by size,
shape, weight, and binding
affinity
Types:
Gel-filtration
chromatography
Ion-exchange
chromatography
Affinity chromatography
Figure 5F Gel-Filtration
Chromatography
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Biochemistry in the Lab
Figure 5G Gel
Electrophoresis
Electrophoresis separates proteins by charge,
molecular weight, and shape
Usually uses agarose or polyacrylamide gels
Protein bands can be visualized with stain or UV
SDS-polyacrylamide gel electrophoresis is widely
used to determine molecular weight
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Biochemistry in the Lab
Figure 5H Mass
Spectrometry
Mass spectrometry uses mass-to-charge ratios and is
a sensitive technique for separating, identifying, and
determining mass
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Biochemistry in the Lab
Protein Sequence-based Function Prediction Once a
polypeptide has been isolated, purified, and
sequenced, determination of function is the next
logical step.
Usually begun with a BLAST (Basic Local Alignment
Search Tool) database search
Protein sequence databases are sufficiently large that
about 50% of queries yield matched sequences close
enough to infer function
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press
Biochemistry in the Lab
Figure 5I X-Ray
Crystallography
X-ray crystallography is used to obtain threedimensional structural information about proteins
Highly ordered crystalline specimens are exposed to an
X-ray beam and the diffraction pattern is used to
construct an electron density map
From McKee and McKee, Biochemistry, 5th Edition, © 2011 by Oxford University Press