index
Announcements
Questions
Review: Titration Curves
Section 4.9: Protein Denaturation and Renaturation
Melting Temperature (T_m)
Anfinsen's Experiment
Section 4.10: Protein Folding
Folding Pathways
Chaperones
Structure of GroEL
Chaperone-Assisted Folding
Section 4.12: Myoglobin and Hemoglobin
Erythrocytes are the Carriers of Hemoglobin
Structure of Myoglobin
Structure of Hemoglobin
Malaria and Human Evolution
Section 4.13: Oxygen Binding
Oxygen Binding Curves
Cooperativity
Structural Changes on Oxygen-Binding
Allosteric Regulation of Hemoglobin
The Bohr Effect
Carbamate Adducts
Questions
References
Next Lecture: Sections 5.1 - 5.3

Fundamentals of Biochemistry

Biochem 380 - Fall 2006

Lecture 012

Outline

Announcements
Questions from previous lecture
Review: Titration Curves
Section 4.9: Protein Denaturation and Renaturation
Section 4.10: Protein Folding and Stability
Section 4.12: Myoglobin and Hemoglobin
Section 4.13: Oxygen Binding

Announcements

Lecture 11 notes have been posted
Quiz 003 scores and answer key have been posted
Reminder: assigned homework problems for Chapter 4: 1, 3, 7, 11, 13, 15
Lecture Schedule has been revised: Chapter 6 moved to next exam, Chapter 20 dropped
Reminder: Midterm 1 is next Thursday, Sept 28, will cover Chapters 1 - 5
Skipping Section 4.11, 4.14
Quiz on Friday will be on Chapter 4, except for sections 6, 7, 8, 11, 14

Questions

Any questions on the material from the previous lecture?
Any problems running the molecular viewing software?

Review: Titration Curves

Inflection points
Acid dissociation constant, pKa
Net charge of a species at a given pH
Isoelectric point (pI)

Section 4.9: Protein Denaturation and Renaturation

After a survey of the elements of protein structure, we will now begin considering the relationship between structure and function
Experiments in the denaturation and renaturation of a protein demonstrate how its function can be lost and regained upon changes in its structure
Denaturation is the disruption of the native conformation of a protein, the characteristic three-dimensional structure that it attains after synthesis
Renaturation is the process in which the native conformation of a protein is reacquired. For many proteins, especially small ones, renaturation can occur quickly and spontaneously under the appropriate conditions
The experiments of Anfinsen in the 1960s on the denaturation and renaturation of ribonuclease A helped to reveal some of the mechanisms and constraints of protein folding and its relationship to function

Melting Temperature (T_m)

Denaturation of proteins usually occurs over a small temperature range, indicating that it is a cooperative process (linked destabilization of interactions)
This is shown at left by the change in several properties of the protein ribonuclease A with temperature, including UV absorbance (blue), viscosity (red) and optical rotation (green)
These changes are associated with an abrupt transition from the native structure of a protein to the denatured state. The midpoint of this transition is the melting temperature, T_m
The T_m of a protein also depends on the pH and ionic strength of a solution. Under physiological conditions the T_m of most proteins is above 50 °C, but for thermophiles it can be much higher

Anfinsen's Experiment

Anfinsen performed a series of experiments on ribonuclease A, a small protein with an activity that could be easily assayed
Instead of temperature, a strong solution of urea was used to denature the protein by destabilizing the structure of water, allowing it to solvate nonpolar groups
2-mercaptoethanol was also required, to reduce the 4 disulfide linkages of the native conformation
The experiment showed that if the disulfide bonds were allowed to form in the denatured state, this would trap the protein in a non-native and inactive conformation (how many possible alternatives?)

Section 4.10: Protein Folding

Anfinsen's experiments provided insight into the alternative pathways that a protein can take in acquiring the native conformation
The details of protein folding pathways are still not well understood, but folding is known to be a rapid process largely driven by hydrophobic interactions that cause collapse of the extended chain through a sequence of intermediate structures
Additional forces such as hydrogen bonding, van der Waals interactions and salt bridges contribute to the final conformation
For many larger proteins, folding and protein stability also require chaperones, molecules that promote formation of the native structure and prevent aggregation and precipitation of misfolded intermediates

Folding Pathways

A large number of possible conformations exist, how is the native (minimal energy) conformation obtained? The search can't be random, because it would take too long (Levinthal's Paradox)
The pathway of folding isn't random, it's determined by a multi-dimensional energy landscape, moving from one energy minimum to the next. The landscape is determined by primary sequence and surrounding environment, sometimes including chaperones

Chaperones

Chaperones are molecules that assist in protein folding. They are especially important for certain kinds of large proteins that take longer to fold and may form inappropriately
Chaperones include the heat shock proteins such as HSP-60 and HSP-70 which are synthesized
These are among the oldest kinds of proteins. We'll look at the structure of GroEl, which is one of the most studied chaperones

Structure of GroEL

GroEl is a large, multi-subunit protein that can accommodate small- and medium-sized proteins
It consists of a central container formed from a stack of rings, and a 'cap' that seals off the folding protein from the external environment
GroEl also has ATP-binding sites that use energy to promote the proper folding and release of the protein

Chaperone-Assisted Folding

Section 4.12: Myoglobin and Hemoglobin

Myoglobin and hemoglobin are proteins that evolved to carry oxygen in the blood streams of vertebrate organisms. This was an important innovation that enabled a reduced viscosity compared to circulatory systems in which oxygen carriers where directly dissolved in the blood plasma
The red color of blood results from the oxygenated forms of myoglobin and hemoglobin, which is caused by a change in the electrical state of the bound heme prosthetic group
Myoglobin is a small monomeric molecule that acts as a secondary carrier of oxygen in the muscle tissue. Hemoglobin is the system-wide carrier of oxygen that is transported through the blood by red blood cells (erythrocytes)

Erythrocytes are the Carriers of Hemoglobin

Mature red blood cells are called erythrocytes. They are biconcave disks that are flexible and smaller than most other cells, allowing them to pass through small capillaries to deliver oxygen throughout the body
A typical human erythrocyte contains around 3 x 10⁸ hemoglobin molecules.
Mammalian erythrocytes lack a nucleus and other cellular organelles, in order to maximize their capacity for carrying hemoglobin. Consequently, they have a limited lifespan of around 3 months, and must be continually replenished through stem cell division and differentiation

Structure of Myoglobin

Myoglobin is largely α-helical in structure, with a total of 8 α-helices. The heme group is bound in a hydrophobic cleft and held there by hydrophobic and van der Waals interactions and hydrogen bonds

Structure of Hemoglobin

Hemoglobin is a tetramer consisting of two α subunits and two β subunits, each of which is similar to myoglobin in structure.
Each subunit has a non-covalently bound heme group, and all 4 subunits are packed together where certain residues of one subunit can interact at the interface with residues of adjacent subunits

Malaria and Human Evolution

Malaria is one of the most widespread diseases afflicting humans. The worst malarial parasite causes over 500 million episodes of disease each year and kills more than one million children each year in Africa alone
Because of its prevalence, malaria is believed to be the strongest source of selective pressure on evolution of the human genome, contributing to sickle-cell disease, thalessemia, G6P deficiency and other erythrocyte defects (most common Mendelian diseases)
Development of effective vaccines against malaria will require further research into interactions with the immune system, genetics, and globin synthesis, function and metabolism

Section 4.13: Oxygen Binding

The binding of oxygen to myoglobin and hemoglobin occurs at the heme group that is noncovalently attached to the peptide chain
The heme group contains a porphoryin ring with a ferrous iron atom bound at the center in the reduced (2+) state. When oxygen binds, the iron is partially oxidized and moves up into the plane of the ring. This partial oxidation allows for the reversible binding of oxygen, and is accompanied by conformational changes in the protein structure through movements of two histidine residues

Section 4.13: Oxygen Binding Curves

Myoglobin, as a single molecule, has a simple hyperbolic binding curve for oxygen, and has a P₅₀ value of only 2.8 torr

In contrast, the multi-subunit nature of hemoglobin reduces its affinity at low concentrations of oxygen and has a much higher P₅₀ value of 26 torr

This increases the dynamic range of hemoglobin, allowing it bind oxygen at high efficiency in the lungs and unload it with good efficiency in the tissues

This difference in binding is due to the cooperative interactions between the hemoglobin subunits

Cooperativity

One analogy that may help to understand the cooperative interactions between subunits is the construction of a dome tent
The insertion of the first tent pole in the collapsed state is always the most difficult. However, after one or two poles are inserted, the global structure of the tent begins to change, making insertion of the remaining poles much easier
In the same way, binding of the first oxygen molecule at one site of hemoglobin promotes a change in structure from the deoxy (T) state to the oxy (R) state that is more favorable for the binding of oxygen at the remaining sites

Structural Changes on Oxygen-Binding

The animation above shows the global, concerted change in structure of the hemoglobin molecule upon oxygen binding
Local conformational changes at all 4 of the subunits are linked, producing the cooperative effect that increases the effectiveness of oxygen unloading

Allosteric Regulation of Hemoglobin

Allosteric regulation is an important mechanism of hemoglogin and is also used extensively in the control of enzyme activity. Allosteric means 'other site' and refers the the effect of small molecules that bind at sites other than the active site of the molecule
In hemoglobin, the molecule 2-3-bisphosphoglycerate (2,3BPG) is a negatively-charged allosteric effector that binds to a positively-charged central region, stabilizing hemoglogin in the T state and promoting unloading of oxygen in tissues

The Bohr Effect

Another regulatory mechanism in hemoglobin is the Bohr effect. This is the effect of pH on the binding affinity of oxygen
The mechanism consists of the protonation of several groups in hemoglobin at low pH. The resulting positive charges of these groups creates salt bridges that stabilize the molecule in the deoxygenated (T) state
The lower pH in tissues is a natural consequence of respiration, and so the Bohr effect helps to promote the unloading of oxygen where it is most needed

Carbamate Adducts

An additional mechanism that contributes to the efficiency of respiration is the ability of CO₂ to form carbamate adducts with the N-terminal chains of hemoglobin
Most CO₂ is transported as dissolved bicarbonate ions, but at high concentrations additional amounts can be attached to hemoglobin.
After being delivered from the tissues to the lungs, the lower concentration of CO₂ favors the reverse reaction, freeing the CO₂ from hemoglobin for release from the lungs

Questions

Questions about the material covered today?

References

GroEL-mediated protein folding: making the impossible, possible,
Lin and Rye, Crit Rev Biochem Mol Biol. 2006 Jul-Aug;41(4):211-39
(A recent review on how the GroEl chaperonin contributes to protein folding)
How Malaria Has Affected the Human Genome and What Human Genetics Can Teach Us about Malaria,
Dominic P. Kwiatkowski, Am. J. Hum. Genet. 77:171�190, 2005
The Malaria System, Science, Oct 2002
http://www.sciencemag.org/feature/plus/sfg/resources/res_malaria.dtl
(Functional genomics of anopheles, plasmodium and humans)

Next Lecture: Sections 5.1 - 5.3

Read Sections 5.1 - 5.3

Table of Contents

Fundamentals of Biochemistry

Biochem 380 - Fall 2006

Lecture 012

Outline

Announcements

Questions

Review: Titration Curves

Section 4.9: Protein Denaturation and Renaturation

Melting Temperature (Tm)

Anfinsen's Experiment

Section 4.10: Protein Folding

Folding Pathways

Chaperones

Structure of GroEL

Chaperone-Assisted Folding

Section 4.12: Myoglobin and Hemoglobin

Erythrocytes are the Carriers of Hemoglobin

Structure of Myoglobin

Structure of Hemoglobin

Malaria and Human Evolution

Section 4.13: Oxygen Binding

Section 4.13: Oxygen Binding Curves

Cooperativity

Structural Changes on Oxygen-Binding

Allosteric Regulation of Hemoglobin

The Bohr Effect

Carbamate Adducts

Questions

References

Next Lecture: Sections 5.1 - 5.3

Melting Temperature (T_m)