Biochemistry Lecture 5

Biochemistry: The Three-Dimensional Structure of Proteins

Chapter Outline

• (4-1) Protein structure and function

• (4-2) Primary structure of proteins

• (4-3) Secondary structure of proteins

• (4-4) Tertiary structure of proteins

• (4-5) Quaternary structure of proteins

• (4-6) Protein-folding dynamics

Tertiary Structure Summary

• The tertiary structure of a protein is defined as the specific 3D shape that the protein folds into, primarily determined by interactions between amino acid R groups.

• Key interactions include:

  • Van der Waals forces:

  • Weak, non-covalent interactions between neutral molecules.

  • Essential for protein structure and function, especially during protein folding.

  • They occur between non-polar (hydrophobic) amino acid side chains which cluster together in the interior, thereby stabilizing the protein's overall shape.

  • Creates a thermodynamically favorable environment by shielding hydrophobic regions from aqueous surroundings.

Learning Check

Question 1:

What type of interaction would you expect between the R groups of the following amino acids in a tertiary structure?

• a. phenylalanine and valine

• b. cysteine and cysteine

Solution 1:

• a. Phenylalanine and valine: Hydrophobic interaction (both have nonpolar R groups).

• b. Cysteine and cysteine: Form a disulfide bond (cysteine residues bond together).

Determination of Tertiary Structure 1

• X-ray crystallography:

  • A technique for determining the 3D atomic and molecular structure of a crystal.

  • Procedure involves:

  • Use of a pure, well-formed crystal exposed to X-rays.

  • Diffraction pattern produced when X-rays scatter off electrons in atoms.

  • Scattered X-rays can either reinforce or cancel each other.

  • Each molecule has a characteristic diffraction pattern.

  • Data analyzed using mathematical techniques (Fourier series).

X-ray Diffraction Steps

• X-rays are directed at a protein crystal.

• Diffraction pattern is measured and transformed into an electron density map.

• This helps to build an atomic model of the protein structure, which can then be evaluated and submitted to the Protein Data Bank (PDB).

Determination of Tertiary Structure 2

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Utilizes a strong magnetic field and radio waves to probe the magnetic properties of atomic nuclei (e.g., 1H, 15N, and 13C) to outline protein structure and dynamics.

  • 2-D NMR collects extensive data points for computer analysis.

  • Data processed using Fourier series and requires substantial computational power as well as milligram quantities of protein in an aqueous solution.

  • Often used to complement X-ray crystallography.

Example: Myoglobin

• Myoglobin features a single polypeptide chain of 153 amino acid residues and incorporates a heme in a hydrophobic pocket.

• Characteristics:

  • Compact structure

  • Eight α-helices

  • Lacks β-pleated sheet regions

  • Exterior predominantly polar side chains

  • Interior primarily nonpolar side chains

• Function: Stores and transports oxygen in muscle tissue, contributing to the red color of meat and aiding muscle function.

Structure of Myoglobin

• The heme group includes:

  • A metal ion, Fe(II), with six coordination sites forming six bonds.

  • Five sites are occupied by nitrogen atoms, and O2 binds at the sixth site.

  • The organic component is protoporphyrin IX, comprising four pyrrole rings linked by bridging groups to form a planar porphyrin ring.

Oxygen Binding Dynamics

• More than one molecule can bind to heme, such as O2 and CO.

• CO Poisoning:

  • CO has a 25,000x greater affinity for heme than O2.

  • Structural dynamics:

  • CO binds in a linear configuration, resulting in a stable interaction.

  • O2 binds at an angle, making it less stable.

  • Binding sites for CO and O2 differ, with histidine E7 forcing CO into a bent configuration, weakening its bond with iron.

Learning Check

Question 2:

The location of heme groups is shown in this level of structure:

• A. primary structure

• B. secondary structure

• C. tertiary structure

• D. quaternary structure

• E. All of these

Solution 2:

E. All of these.

Hemoglobin Structure

• Hemoglobin is a globular protein that transports oxygen in blood, consisting of four polypeptide chains:

  • Two α-chains (141 amino acids each)

  • Two β-chains (146 amino acids each)

• The quaternary structure of hemoglobin is significant, as it consists of multiple subunits held together by stabilizing interactions typical of tertiary structures.

Oxygen Binding and Cooperativity

• The structure of oxygenated hemoglobin differs from deoxygenated hemoglobin, with less room at the center.

• Binding of O2 demonstrates positive cooperativity; as one O2 binds, the structure alters, facilitating subsequent O2 bindings.

Protein Structures Summary

• Proteins exhibit primary, secondary, tertiary, and often quaternary structural levels.

Chemistry Link to Health: Sickle-Cell Anemia

• Caused by an abnormality in hemoglobin structure where polar glutamic acid is replaced by nonpolar valine.

• Effects:

  • Nonpolar valine R group attracts nonpolar regions within beta hemoglobin chains, altering the shape of red blood cells from rounded to crescent (sickle) shaped.

  • This prevents effective oxygen transport.

  • Sickle-cell hemoglobin forms insoluble fibers that clog capillaries, causing inflammation and organ damage due to low oxygen levels.

Myoglobin vs. Hemoglobin

2,3-Bisphosphoglycerate Binding to Hemoglobin

• Binding is electrostatic and decreases hemoglobin's oxygen-binding capacity, crucial for maintaining oxygen supply to the fetus.

• Produced during glycolysis; it reduces hemoglobin's affinity for oxygen, facilitating oxygen release to tissues.

Cytochrome C

• Cytochrome c is a small hemeprotein located in the inner mitochondrial membrane, critical for cellular respiration and apoptosis.

• Its structure contains a porphyrin ring resembling myoglobin and hemoglobin.

Learning Check

Question 3:

The protein myoglobin:

• A. contains a high degree of β-pleated sheet structure

• B. carries oxygen in the bloodstream

• C. contains no histidine

• D. contains a heme group

Solution 3:

D. contains a heme group.

Denaturation of Proteins 1

• Denaturation occurs when stabilizing interactions among residues that form secondary, tertiary, or quaternary structures are disrupted, resulting in biological inactivity without affecting amide bonds among amino acids.

Denaturation of Proteins 2

• Loss of secondary and tertiary structure arises from:

  • Increased temperature

  • Extreme pH (acidic or basic)

  • Introduction of certain organic compounds or heavy metal ions

  • Mechanical agitation

• Consequences:

  • Unfolding of globular proteins

  • Disruption of tertiary structure

  • Loss of biological activity.

Denaturation by Heat

• Proteins denatured at temperatures above 50ºC.

• Heat disrupts hydrogen bonds and hydrophobic interactions among nonpolar residues.

• Note: Does not alter nutritional value but enhances digestibility.

• Example: Autoclaving surgical items sterilizes by denaturing bacterial proteins.

Denaturation by Acids and Bases

• Changing pH leads to protein denaturation by:

  • Breaking hydrogen bonds

  • Disrupting ionic bonds and salt bridges.

• Example: Tannic acid in burn ointments causes protein coagulation, creating a protective layer and reducing fluid loss.

Denaturation by Organic Compounds

• Alcohols such as ethanol and isopropyl alcohol act as disinfectants by replacing protein's hydrogen bonds with their own and disrupting internal hydrogen bonding.

• Example: Alcohol swabs are used to prepare skin for injections due to their ability to permeate bacterial cell walls and coagulate internal proteins.

Denaturation by Heavy Metal Ions

• Heavy metal ions (Ag+, Pb2+, Hg2+) denature proteins by bonding with ionic residues or reacting with disulfide bonds.

• Example: AgNO3 is used in newborns' eyes to eliminate bacteria causing gonorrhea.

Denaturation by Mechanical Agitation

• Whipping cream and egg whites applies mechanical agitation that stretches polypeptide chains and disrupts stabilizing interactions.

Denaturation Summary

Learning Check

Question 4:

Tannic acid is used to form a scab on a burn. An egg is hard-boiled by placing it in boiling water. What is similar about these two events?

Solution 4:

Both acid and heat cause denaturation of proteins, breaking bonds in secondary, tertiary, and quaternary structures.

Learning Check

Question 5:

What happens when a protein is denatured?
A. Its secondary structure is disrupted but its primary structure remains intact.
B. Its primary structure is disrupted but its secondary structure remains intact.
C. It is broken apart into its constituent amino acids.
D. It becomes all α-helix.

Solution 5:

A. Its secondary structure is disrupted but its primary structure remains intact.

Additional Information: Denaturation and Refolding

• β-mercaptoethanol: Utilized to break disulfide bridges in tertiary and quaternary structures.

• Urea: Added to facilitate unfolding and increase accessibility of disulfides to reducing agents.

• Native conformations can potentially be recovered by removing both mercaptoethanol and urea.

The Importance of Proper Protein Folding

• In the cellular environment, proteins may fold incorrectly or associate with others before completing their folding process.

Protein-Folding Chaperones

• Molecular chaperones assist in correct and timely protein folding.

• Examples:

  • hsp70: The first discovered chaperone, highly conserved across organisms from prokaryotes to humans.

  • α-hemoglobin stabilizing protein (AHSP): Prevents α-chain damage to blood cells and assists in delivering α-chains to β-chains.

Predicting Protein Structure Using Bioinformatics

• Bioinformatics aids in searching databases for known structures to establish sequence homology.

• Methods used include:

  • Fold recognition from databases

  • De novo prediction based solely on amino acid sequences

  • Modeling algorithms

Role of Hydrophobic Interactions in Protein Folding

• Hydrophobic interactions are significant drivers in the protein folding process.

• Folding organizes so that:

  • Nonpolar hydrophobic side chains are located internally, away from water

  • Polar side chains are positioned outside, interacting with the aqueous environment.

• Example: Liposomes – spherical aggregates of lipids arranged with polar head groups in contact with water while nonpolar tails are sheltered from water.

Figure 4.34 - Schematic Diagram of a Liposome

• Illustrates the three-dimensional structure with hydrophilic head groups aligned with the aqueous environment and hydrophobic tails oriented inward, away from water.