Amino Acids and Proteins Notes

Introduction to Proteins

• Proteins are nitrogenous substances found in the protoplasm of all animal and plant cells.
• They constitute approximately 20% of the human body.
• Proteins are large, complex molecules essential for normal cell functioning.
• They are composed of amino acids linked by peptide bonds, forming long chains.
• Proteins primarily contain carbon, hydrogen, oxygen, nitrogen, and sulfur. Some may also contain phosphorus or iron (e.g., nucleoproteins and hemoglobin).

Functions of Proteins

• Catalysts: Enzymes that speed up biochemical reactions.
• Transport/Storage: Transport or store molecules (e.g., oxygen).
• Structural Support: Provide mechanical support.
• Immune Protection: Antibodies for immune defense.
• Movement Generation: Enable movement.
• Nerve Impulse Transmission: Transmit nerve impulses.
• Growth and Differentiation Control: Regulate growth and differentiation.

Amino Acids: The Building Blocks

• Amino acids are the building blocks of proteins.
• Proteins are natural polymers of amino acids, ranging from two to thousands in number.
• Amino acids contain nitrogen, carbon, hydrogen, and oxygen.
• Key properties of amino acids include size, shape, charge, polarity, hydrophobicity, aromaticity, conformation, propensity to adopt specific conformations, and relative position in proteins.

Types of Amino Acids

• Non-polar: Alanine (Ala/A), Valine (Val/V), Leucine (Leu/L), Isoleucine (Ile/I), Tryptophan (Trp/W), Methionine (Met/M), Phenylalanine (Phe/F).
• Polar Uncharged: Serine (Ser/S), Threonine (Thr/T), Tyrosine (Tyr/Y), Asparagine (Asn/N), Glutamine (Gln/Q).
• Electrically Charged (Acidic): Aspartate (Asp/D), Glutamate (Glu/E).
• Electrically Charged (Basic): Lysine (Lys/K), Arginine (Arg/R), Histidine (His/H).

Special Amino Acids

• Proline: A unique amino acid where the side chain connects to the protein backbone twice, forming a five-membered nitrogen-containing ring. It is a secondary amino acid and an imino acid.
• Cysteine and Cystine: Cysteine has a sulfur group. Two cysteines can form a disulfide bond, creating cystine.

Chirality of Amino Acids

• Amino acids are chiral molecules, meaning they can exist in two versions that are mirror images of each other (chirality).
• A chiral center is a carbon atom with four different groups attached.
• Chiral molecules have right-handed and left-handed versions, called enantiomers.
• Organisms on Earth use left-handed amino acids to make proteins (homochirality).
• D and L configurations refer to the absolute configuration relative to glyceraldehyde. Only L-amino acids are constituents of proteins.

R-S Configuration

• The R-S configuration is assigned by ranking the four groups attached to the chiral center based on atomic number using the Cahn-Ingold-Prelog priority rules.
• Glycine is the only achiral amino acid as its R group is hydrogen.

Peptide Bonds

• Almost all peptide bonds in proteins are trans due to steric hindrance in the cis configuration.
• Steric hindrance refers to repulsive forces between overlapping electron clouds.

Essential Amino Acids

• Essential amino acids (EAAs) cannot be synthesized in animal cells and must be obtained through diet.
• Plant proteins may be incomplete sources, with cereals lacking lysine and legumes lacking methionine.
• A diet combining cereals and legumes can provide adequate amounts of these key amino acids.
• Quinoa contains good amounts of both lysine and methionine.

Amphoteric Nature of Amino Acids

• Amino acids are amphoteric, exhibiting both acidic (COOH) and basic (NH2) properties.
• Their structure is pH-dependent due to the presence of dissociable protons.
• Ampholytes are molecules that are amphoteric.

Acid Dissociation Constant (Ka) and pKa

• Ka is a quantitative measure of the strength of an acid in solution.
• pKa = -log(Ka)
• Acetic acid (pKa = 4.76) is a weak acid, while hydrochloric acid (HCl) (pKa = -8.3) is a strong acid.

Henderson-Hasselbalch Equation

• The acid dissociation constant (Ka) distinguishes strong acids from weak acids.
• pKa = pH at which 50% of a substance is ionized.

pKa in Amino Acids

• pKa determines the protonation state of amino acids and the charges of functional groups.
• Amino acids contain at least two functional groups: an amino group (NH2) and a carboxyl group (COOH).
• The pKa values of these groups determine the pH at which they are protonated or deprotonated.

Ionization States

• Amino group (NH2) pKa is typically 9-10: below this pH, it is protonated (NH3+); above, it is deprotonated (NH2).
• Carboxylic group (COOH) pKa is typically 2-3: below this pH, it is protonated (COOH); above, it is deprotonated (COO-).
• At physiological pH (7.4), the amino group is protonated (NH3+), and the carboxylic group is deprotonated (COO-), resulting in a zwitterion.
• A zwitterion can act as either an acid or a base.

Isoelectric Point (pI)

• The isoelectric point (pI) is the pH at which a molecule carries no net electric charge.
• pI =
  onumber\frac{1}{2} (pK1 + pK2)
• For amino acids, pK1 corresponds to pKα-COOH, and pK2 corresponds to pKα-NH2.
• The pI is the average of the pKa values of the ionizable groups that have a positive and negative charge.
• For glycine (no side chain), the pI is approximately 6.0.
• For histidine (ionizable side chain), the pI is approximately 7.6.

Titration of Glycine

• Most amino acids are similar to glycine in their ionization properties.
• Exceptions include Glu, Asp, Lys, Arg, and His, which have polar, charged side chains.
• At a very low pH, glycine is fully protonated (COOH and NH3+).
• As OH- is added, H+ is removed from COOH first (pH ≈ 2), then from NH3+ (pH ≈ 10).
• pK1 is for the loss of a proton from the carboxyl group, and pK2 is for the loss of a proton from the amino group.

pKa Values of Amino Acids with Ionizable R-Groups

• Ionizable amino acids contain nitrogen or oxygen in their side chains.
• pKaR (R group) influences the shape of the titration curve and new properties of these amino acids.
• Alpha carboxylic acids ionize at acidic pH (pKa < 6), and alpha amino groups ionize at basic pH (pKa > 8).
• When titrating a fully protonated amino acid, the alpha carboxylic acid loses a proton first, followed by the amino group.

pI for Acidic and Basic Amino Acids

• For Aspartic acid and Glutamic acid:
  • pK1 < pKR < pK2
  • Possible charges: +1, 0, -1, -2
  • pI =
    onumber\frac{pK1 + pKR}{2}
• For Lysine, Arginine, and Histidine:
  • For Lys and Arg:pK1 < pK2 < pKr
  • For His: pK1 < pKr < pK2
• Titration of Lys:
  • Possible charges: +2, +1, 0, -1
  • pI =
    onumber\frac{pK2 + pKr}{2} =
    onumber\frac{9.2 + 10.8}{2} = 10
• Titration of His:
  • Possible charges: +2, +1, 0, -1
  • pI =
    onumber\frac{pK1 + pKR}{2} =
    onumber\frac{6 + 9.3}{2} = 7.6

Calculating pI for Dipeptides

1. Draw the peptide at its most protonated form (low pH).
2. Calculate the overall charge.
3. Calculate the change in charge as pH rises.
4. Use two pKa values surrounding the peptide at zero charge.

• pI =
  onumber\frac{PK1 + pK2}{2}

UV Spectrums (Spectra) of Amino Acids

• A spectrum is obtained by measuring the absorption of light as a function of its frequency or wavelength.
• Techniques include absorption spectroscopy (UV-Vis) and emission spectroscopy (fluorescence).

Lambert-Beer Law

• When incident light (I0) passes through a medium with concentration C, part of the light is absorbed.
• A = εCl, where:
  • A is absorbance.
  • ε is the molar absorption coefficient (L/mol·cm).
  • C is the concentration.
  • l is the path length.

UV-Visible Spectroscopy

• UV-Vis spectroscopy measures the amount of UV or visible light absorbed or transmitted through a sample.
• The UV-visible range covers the wavelength range of 150-800 nm.
• Absorption spectroscopy is carried out in clear/transparent solutions.

Components of Spectrophotometry

1. Source (UV or visible light).
2. Wavelength selector (monochromator).
3. Sample container.
4. Detector.
5. Signal processor and readout.

Chromophores in Proteins

• Chromophores are chemical groups that absorb light at specific frequencies.
• Examples include tryptophan, tyrosine, and phenylalanine.

UV-Visible Absorbance

• Near UV region: 250-500 nm
• Far UV region: 190-250 nm
• Vacuum UV region: <190 nm
• The chromophoric parts of Trp, Tyr, and Phe are responsible for the near UV region with maximum absorption around 280 nm.
• Disulfide bonds between cysteine residues show an absorbance band near 260 nm.
• Peptide bonds are the major chromophore in the far UV region, with a strong absorption band around 190 nm and a weak band around 210-220 nm.
• Applications:
  • Structural analysis
  • Quantification
  • Interaction with other molecules
  • Enzymatic activity

Proteins: Composition, Structure, and Function

• Proteins yield amino acids upon hydrolysis and are the basis for major structural components of animal and human tissues.
• They act as biological catalysts (enzymes), form structural parts of organisms, act as molecules of immunity, and provide fuel.
• Protein molecules contain nitrogen, carbon, hydrogen, and oxygen.

Classification of Proteins

• Simple Proteins: Albumins, Globulins, Globins, Prolamines, Histones, Protamines, Albuminoids.
  • Albumins: Water-soluble, coagulated by heat (e.g., albumin, lactalbumin, ovalbumin).
  • Globulins: Insoluble in water, soluble in dilute salt solutions (e.g., lactoglobulins, immune globulins, myosin).
  • Globins: Rich in histidine and unite with heme to form hemoglobin.
  • Prolamines: Soluble in 70-80% ethanol, insoluble in water (e.g., gliadin of wheat and zein of maize).
  • Histones: Soluble in water but not in ammonium hydroxide, present in the nucleus.
  • Protamines: Like histones but present in sperm cells, soluble in ammonium hydroxide.
  • Albuminoids: Also called scleroproteins (e.g., collagen, elastin, and keratin).
• Conjugated Proteins: Contain a non-protein group (prosthetic group) attached to a protein part.
  • Nucleoproteins (Protein + Nucleic acid).
  • Chromoproteins (Protein + colored prosthetic group, e.g., chlorophyll and hemoglobin).
  • Glycoproteins (Protein + Carbohydrate).
  • Phosphoproteins (Protein + Phosphate group).
  • Lipoproteins (Protein + Lipid group).
  • Metalloproteins (Protein + Metals).
• Derived Proteins: Degradation products obtained by acids, alkalis, and enzymes.
  • Primary Proteins: Insoluble in water, soluble in acids and alkalis. Formed by slight changes in the protein molecule.
    • Proteans: Insoluble products formed by water, dilute acids, and enzymes. e.g., myosan from myosin, fibrin from fibrinogen.
    • Metaproteins: Formed by the action of acids and alkalis upon protein. Insoluble in neutral solvents.
    • Coagulated proteins: Insoluble products formed by the action of heat or alcohol on natural proteins, e.g., cooked meat and cooked albumin.
  • Secondary Proteins: Soluble in water and coagulated by heat. Formed in the progressive hydrolytic cleavage of the peptide bonds of the protein molecule.

Protein Structure

• Four levels of structure: primary, secondary, tertiary, and quaternary.

Primary Structure

• Linear sequence of amino acids held together by covalent bonds.
• Å = 10^{-10} m
• Peptide bond formation is a condensation reaction or dehydration synthesis.
• Peptides have direction (amino-terminal to carboxyl-terminal).

Secondary Structure

• Local folded structures, such as alpha-helices and beta-sheets.
• Alpha-Helix:
  • All NH and CO groups are hydrogen-bonded, except at the ends.
  • Dense structure with no space inside.
  • Proline is a helix breaker.
  • Right-handed twist (alpha-R).
  • R-groups point out of the helix.
  • The Helix has 3.7 Amino acids per turn
  • Rise: distance between two amino acids
  • Pitch
• Beta-Pleated Sheet:
  • Can be parallel or antiparallel.
  • Pleated shape due to the tetrahedral α-carbon.
  • R groups alternate above and below the sheet.
• Beta-Turns (Reverse Turns):
  • Common structural elements that reverse the direction of polypeptide chains.
  • Hydrogen bonds between the carbonyl of residue i and the N-H group of residue i+3.
  • Longer forms of turns are called loops or omega loops.

Tertiary Structure

• Overall three-dimensional arrangement of all atoms in a protein.
• Interacting segments are held in position by non-covalent interactions and disulfide bonds.
• Residues with hydrophobic side chains tend to be found in the interior, while hydrophilic side chains are on the surface.
• Disulfide bonds (covalent linkages between cysteine residues) stabilize tertiary structure.

Quaternary Structure

• Arrangement of multiple polypeptide chains (subunits) in three-dimensional complexes.
• Held together by hydrogen bonds and covalent bonds (disulfide bridges).
• Example: Hemoglobin (binds oxygen via heme groups).
• Example: Porin (transmembrane protein with hydrophobic exterior and hydrophilic interior).

Major Protein Types

Fibrous Proteins

• Long and spindly, with primarily primary and secondary structures.
• Little or no tertiary structure.
• Long parallel polypeptide chains with cross-linkages.
• Mostly insoluble.
• Structural roles.
• Repetitive amino acid sequences.
• Less sensitive to temperature and pH changes.
• Examples: Keratin, Collagen, Silk.

Globular Proteins

• Highly folded and compact with complex tertiary and quaternary structures.
• Folded into spherical/globular shapes, soluble in water.
• Most non-polar groups are inside, and polar groups stay outside.
• Irregular amino acid sequences.
• More sensitive to temperature and pH.
• Examples: Hemoglobin, Enzymes, Hormones (Insulin), Antibodies.

Conformational Stability and Denaturation

• Conformation: Folded 3D structure (active form).
• Denaturation: Protein unfolds or becomes inactive.
• Factors contributing to stability:
  • Primary structure: Peptide bonds.
  • Secondary structure: Backbone interactions held together by hydrogen bonds.
  • Tertiary structure: Distant interactions between groups (Van der Waals, hydrophobic interactions, disulfide bonds).
  • Quaternary structure: Interactions between individual protein subunits.
  • Solvation shell: The layer of solvent surrounding a protein. Water is a polar molecule and there is a electronegative oxygen atom with a predominantly negative charge leaving the positive charge over next to the hydrogen atom. The electronegative Oxygen atoms are stabilizing all the positively charged amino acid residues on the exterior of this protein.
• Denaturation: Process in which a protein loses its native shape and becomes biologically inactive.
• Denaturing agents:
  • Temperature: Increased kinetic energy disrupts bonds.
  • pH: Alters electrostatic interactions.
  • Chemicals: Disrupt hydrogen bonding.

• Consequences of denaturation: reduced solubility, decreased biological activity, altered shape, increased reactivity, susceptibility to enzymatic hydrolysis.

Denaturation of Proteins

• Results in reduced solubility, decreased biological activity, loss of crystallization properties, increased reactivity, altered shape, and susceptibility to hydrolysis.

Strange, Extremely Disordered Proteins

• Newly discovered group of proteins with unusual shapes and abilities to protect against protein clumps associated with neurodegenerative diseases.
• Heat-resistant and widespread in animals.
• Long, flexible string-like structure.

Hydrolysis of Proteins

• Peptide bonds are broken through hydrolysis reactions.
• Occurs in the stomach with enzymes catalyzing the breakdown of proteins into amino acids.
• Primary structure breaks up by breaking covalent peptide bonds.

Enzymes

• Biological catalysts that speed up biochemical reactions.
• Most enzymes are globular proteins (tertiary and quaternary structure).

Structure of Enzymes

• Active site: region that binds substrates, cofactors, and prosthetic groups.
• Active sites occupy less than 5% of the total surface area.
• Specific shape due to tertiary structure.
• High specificity for substrates.

Components of active sites

• Binding site: chooses the substrate and binds it to active site
• Catalytic site: It performs the catalytic action of enzyme

Enzyme Classification

• Systematic classification by the International Enzyme Commission.
• Based on the type of reactions catalyzed.

Enzyme Commission Number (EC Number)

• Numerical classification scheme for enzymes.
• Developed by the International Union of Biochemistry and Molecular Biology (IUBMB).
• Consists of four digits indicating different aspects of the enzyme's classification.
• Example: Hexokinase (EC 2.7.1.1).

Major Enzyme Classes:

1. Oxidoreductases: Catalyze redox reactions.
2. Transferases: Catalyze the transfer of functional groups.
3. Hydrolases: Catalyze hydrolysis reactions.
4. Lyases: Cleave covalent bonds without water or oxidation.
5. Isomerases: Catalyze intramolecular changes.
6. Synthetases: Catalyze the joining of two molecules.