Protein Structure and Function Notes

Protein Structure/Function

Proteins: Diverse Macromolecules

Proteins are essential to cellular function and are incredibly diverse macromolecules.
Examples of proteins include:
- Carrier protein HPr
- Hemoglobin
- DNA
- Lysozyme
- Catalase
- Myoglobin
- Deoxyribonuclease
- Collagen
- Cytochrome c
- Porin
- Chymotrypsin
- Calmodulin
- Insulin
- Alcohol dehydrogenase
- Aspartate transcarbamoylase

Amino Acids: Building Blocks of Proteins

Amino acids are the basic building blocks of proteins.
The general formula of an amino acid is:
$H_2N-C(H)(R)-COOH$
R represents one of 20 different side chains.
At pH 7, both the amino and carboxyl groups are ionized.
The ionized form is:
$H_3N^+-C(H)(R)-COO^-$

Peptide Bonds

Amino acids are joined by peptide bonds.
The amine of one amino acid is covalently bonded to the carboxyl carbon of a second amino acid through a condensation reaction.
The polypeptide backbone is a consistent and repeating sequence.

Polypeptide Backbone and Side Chains

Proteins (polypeptides) consist of a regular polypeptide backbone and a variety of side chains.
The polypeptide backbone has an amino terminus (N-terminus) and a carboxyl terminus (C-terminus).
Examples of amino acids with their side chains:
- Methionine (Met)
- Aspartic acid (Asp)
- Leucine (Leu)
- Tyrosine (Tyr)

Twenty Amino Acids Grouped by Side Chains

There are twenty amino acids, grouped according to their side chains:
- Negatively charged (acidic):
  - Aspartic acid (Asp, D)
  - Glutamic acid (Glu, E)
- Positively charged (basic):
  - Arginine (Arg, R)
  - Lysine (Lys, K)
  - Histidine (His, H)
- Uncharged polar:
  - Asparagine (Asn, N)
  - Glutamine (Gln, Q)
  - Serine (Ser, S)
  - Threonine (Thr, T)
  - Tyrosine (Tyr, Y)
- Nonpolar:
  - Alanine (Ala, A)
  - Glycine (Gly, G)
  - Valine (Val, V)
  - Leucine (Leu, L)
  - Isoleucine (Ile, I)
  - Proline (Pro, P)
  - Phenylalanine (Phe, F)
  - Methionine (Met, M)
  - Tryptophan (Trp, W)
  - Cysteine (Cys, C)

Acidic Side Chains

Examples:
- Aspartic acid (Asp, D): $H-CH_2-C(=O)-O^-$
- Glutamic acid (Glu, E): $H-CH2-CH2-C(=O)-O^-$

Basic Side Chains

Examples:
- Lysine (Lys, K): $-CH2-CH2-CH2-CH2-NH_3^+$
- Arginine (Arg, R): $-CH2-CH2-CH2-NH-C(NH2)_2^+$
 - The positive charge is stabilized by resonance.
- Histidine (His, H): $-CH_2-\text{imidazole ring}$
 - The nitrogens have a relatively weak affinity for H+ and are only partly positive at neutral pH.

Polar Side Chains

Examples:
- Asparagine (Asn, N): $-CH2-C(=O)NH2$
- Glutamine (Gln, Q): $-CH2-CH2-C(=O)NH_2$
- Serine (Ser, S): $-CH_2-OH$
 - The -OH group is polar.
- Threonine (Thr, T): $-CH(OH)-CH_3$
- Tyrosine (Tyr, Y): $-CH_2-\text{phenol ring}$

Nonpolar Side Chains

Examples:
- Alanine (Ala, A): $-CH_3$
- Valine (Val, V): $-CH(CH3)2$
- Methionine (Met, M): $-CH2-CH2-S-CH_3$
- Tryptophan (Trp, W): $-CH_2-\text{indole ring}$
- Leucine (Leu, L): $-CH2-CH(CH3)_2$
- Isoleucine (Ile, I): $-CH(CH3)-CH2-CH_3$
- Glycine (Gly, G): $-H$
- Proline (Pro, P): cyclic structure (imino acid).
- Phenylalanine (Phe, F): $-CH_2-\text{benzene ring}$
- Cysteine (Cys, C): $-CH_2-SH$
 - Disulfide bonds can form between two cysteine side chains in proteins: $-CH2-S-S-CH2-$

Levels of Protein Structure

Primary
Secondary
Tertiary
Quaternary

Primary Structure

Primary structure is the sequence of amino acids.
Example sequence: MNGQGCELGHSNGDIISQNQQKGWWTIGLINGQHKYMTAETFGFKLNANGASLKKKQLWTLEPSNTGESIIYLRSHLNKYLSVDQFGNVLCESDERDAGSRFQISISEDGSGRWALKNESRGYFLGGTPDKLVCTAKTPGASEFWTVHLAARPQVNLRSIGRKRFAHLSESQDEIHVDANIPWGEDTLFTLEFRAEEGGRYALHTCNNKYLNANGKLQVVCNEDCLFSAEYHGGHLALRDRQGQYLSPIGSKAVLKSRSSSVTRDELFSLEDSLPQASFIAGLNLRYVSVKQGVDVTANQDEVGENETFQLEYDWSAHRWALRTTQDRYWCLSAGGGIQATGNRRCADALFELIWHGDGSLSFRANNGKFLATKRSGHLFATSESIEEIAKFYFYLINRPILVLKCEQGFVGYRTPGNLKLECNKATYETILVERAQKGLVHLKAHSGKYWRIEGESISVDADAPSDGFFLELREPTRICIRSQQGKYLGATKNGAFKLLDDGTDSATQWEF

Cartoon Styles to Display Protein Structure

Backbone model
Wire model
Ribbon model
Space-filling model

Secondary Structure

Secondary structure includes local folding patterns.
Two common folding patterns:
- Alpha Helix
- Beta Sheet
Both are based on hydrogen bonding between N-H and C=O groups in the polypeptide backbone.

Alpha Helix

Polypeptide backbone is at the center
Side chains stick outward
Short helices are very common
Often found in proteins that span the cell membrane
Alpha-helices can coil around one another (coiled-coil)
High tensile strength

Beta Sheet

Hydrogen bonding occurs between strands or segments of the polypeptide
Strands can be parallel or antiparallel
Forms a rigid, pleated sheet
High tensile strength

Tertiary Structure

Tertiary structure refers to the fully folded three-dimensional conformation of a polypeptide.
Proteins may contain alpha-helices, beta-sheets, and unstructured regions.

Noncovalent Interactions Driving Tertiary Structure

Electrostatic attractions (e.g., between aspartic acid (D) and arginine (R))
Hydrogen bonds
Van der Waals attractions
Hydrophobic effect (nonpolar side chains cluster inside)

Protein Folding

Proteins fold to minimize outward exposure of hydrophobic side chains in an aqueous environment.
Polar side chains can form hydrogen bonds to water.
Nonpolar side chains are packed into a hydrophobic core region.

Protein Domains

Protein domains represent a level between secondary and tertiary structure.
They typically highlight a function of a protein.

Quaternary Structure

Quaternary structure is when multiple polypeptides come together to form a single functional protein.
Examples:
- Dimer of the CAP protein
- Tetramer of neuraminidase protein

Larger Assemblies

Quaternary structure can form even larger assemblies like dimers, rings, or helices.

Even Larger Assemblies

Examples of larger assemblies include spherical shells, filaments, and hollow tubes.

Protein Functions

Proteins carry out highly diverse functions, including:
- Enzymes: Catalyze covalent bond breakage or formation.
  - Examples: alcohol dehydrogenase, pepsin, ribulose bisphosphate carboxylase, DNA polymerase, protein kinase
- Structural Proteins: Provide mechanical support to cells and tissues.
  - Examples: collagen, elastin, tubulin, actin, keratin
- Transport Proteins: Carry small molecules or ions.
  - Examples: serum albumin, hemoglobin, transferrin, bacteriorhodopsin, glucose transporters, Ca2+ pump
- Motor Proteins: Generate movement in cells and tissues.
  - Examples: myosin, kinesin, dynein
- Storage Proteins: Store amino acids or ions.
  - Examples: ferritin, ovalbumin, casein
- Signal Proteins: Carry extracellular signals from cell to cell.
  - Examples: insulin, netrin, nerve growth factor (NGF), epidermal growth factor (EGF)
- Receptor Proteins: Detect signals and transmit them to the cell's response machinery.
  - Examples: rhodopsin, acetylcholine receptor, insulin receptor, adrenergic receptor
- Transcription Regulators: Bind to DNA to switch genes on or off.
  - Examples: Lac repressor, DNA-binding proteins
- Special-Purpose Proteins: Highly variable.
  - Examples: antifreeze proteins, green fluorescent protein, monellin, glue proteins

Protein Binding

The activity of proteins depends on their ability to bind to other molecules.
Interactions are highly specific.
Shape is highly important to protein binding.
Interactions are driven by many noncovalent bonds.

Ligands and Binding Sites

A molecule that binds to a protein is called a ligand.
The protein region that associates with a ligand is called a binding site.
Binding sites are usually a cavity in the protein surface.
Common protein domains are involved in ligand binding.

Antibodies

Antibodies are proteins that bind particular target molecules (antigens).
Target binding is tight and specific.
Targets are either inactivated or marked for destruction.
Primary function: defend us from infection.

Antibody Structure

Antibodies consist of four polypeptide chains, representing quaternary structure.
Each antibody has a variable domain and a constant domain.
Antibodies are incredibly useful tools for cell biologists.

Raising Antibodies in Animals

Inject an antigen into an animal (usually multiple injections).
The animal’s B cells make large amounts of antibodies specific to the antigen.
The antibody can be purified from the blood.

Chemical Tagging of Antibodies

Antibodies can be chemically tagged for use in microscopy.

Enzymes as Catalysts

Many proteins are enzymes - highly specific catalysts.
Even favorable reactions require some activation energy.
Catalysts (enzymes) promote a chemical reaction by lowering the activation energy needed for the reaction to occur.
The ligand that binds to an enzyme is called the substrate.
The site of enzyme-substrate interaction is called the active site.

Lysozyme

Lysozyme is an enzyme that cleaves polysaccharide chains.
The reaction that breaks a bond between subunits of a polysaccharide is hydrolysis.
The lysozyme active site is a long groove that holds six sugars of a chain.

Lysozyme Mechanism

Specific amino acid side chains in the active site are critical for enzyme function.
Example: Lysozyme active site with an oligosaccharide substrate.
Hydrolysis of the bond between sugars D and E.
Key amino acids: Glu 35 and Asp 52.

Feedback Inhibition

Regulation of enzyme activity through feedback inhibition.
The product of a metabolic pathway inhibits an enzyme earlier in the pathway.

Allosteric Regulation

Many proteins are allosteric and can adopt multiple conformations.
Activity can be regulated by a shift from one conformation to another.
Binding to a regulatory ligand can induce a conformational shift.
True of many proteins – not just enzymes.
Regulatory ligands can promote or inhibit protein activity.

Protein Phosphorylation

A very common mechanism of regulating protein activity.
Kinases add phosphate groups, and phosphatases remove them.
Process:
- ATP donates a phosphate group to a protein, activating it.
- The phosphorylated protein can then be dephosphorylated by a phosphatase, returning it to its inactive state.

Phosphorylatable Amino Acids

The three amino acid side chains that can be phosphorylated are serine, threonine, and tyrosine.
The common feature in these side chains is the presence of a hydroxyl (-OH) group.

Covalent Modifications of Proteins

There are many more covalent modifications of proteins beyond phosphorylation.
More than 100 types of covalent modifications of proteins are known.
Proteins can be modified and regulated at several sites simultaneously.

ATPases and GTPases

ATPases and GTPases are proteins that hydrolyze nucleotides to regulate activity.
GTP-binding proteins:
- Active when bound to GTP.
- Inactive when bound to GDP.
- Hydrolysis of GTP to GDP inactivates the protein.
- GTP binding reactivates the protein.