Amino Acids: Structures and Properties

Standard Amino Acids

The 20 standard amino acids are the building blocks of proteins. Each has a central (alpha) carbon, an amino group, a carboxyl group, a hydrogen atom, and a unique side chain (R group).

• Three-letter codes and single-letter abbreviations are used to represent amino acids in sequences.

• L-amino acids are the naturally occurring isomers in proteins. The L and D designations refer to the configuration around the alpha carbon, with L being the configuration found in biological systems.

• Fischer projections are used to depict the absolute configuration of amino acids. The D isomer is the mirror image of the L isomer.

Classification by R Groups

Amino acids are classified based on the properties of their side chains:

• Non-polar (hydrophobic): e.g., Alanine, Valine, Leucine

• Polar (uncharged): e.g., Serine, Threonine, Asparagine

• Aromatic: e.g., Phenylalanine, Tyrosine, Tryptophan

• Acidic (negatively charged): e.g., Aspartate, Glutamate

• Basic (positively charged): e.g., Lysine, Arginine, Histidine

Ionization, Titration Curves, and Buffering

• pKa values indicate the pH at which a group is 50% ionized. Each amino acid has at least two pKa values (amino and carboxyl termini); some have ionizable side chains.

• Isoelectric point (pI): The pH at which the amino acid has no net charge.

• Titration curves show the ionization states as a function of pH and are used to determine pI and buffer regions.

• Henderson-Hasselbalch equation:

• Used to calculate the ratio of protonated to deprotonated species and to prepare buffers.

Solubility and pKa Values

• Ionic and polar compounds increase water solubility; non-polar compounds decrease it.

• pKa values differ for free amino acids and those in peptides due to the chemical environment.

Peptide and Protein Isolation and Purification

Precipitation and Chromatography

• Ammonium sulfate precipitation: Exploits differences in protein solubility to separate proteins from solution.

• Ion exchange chromatography: Separates proteins based on charge.

• Size exclusion (gel filtration) chromatography: Separates proteins by size; larger molecules elute first.

• Affinity chromatography: Separates proteins based on specific binding interactions (e.g., with ligands or antibodies).

Electrophoresis Techniques

• SDS-PAGE: Separates proteins by molecular weight. SDS denatures proteins and gives them a uniform negative charge.

• Isoelectric focusing: Separates proteins based on their isoelectric point (pI).

• Two-dimensional electrophoresis: Combines isoelectric focusing and SDS-PAGE for high-resolution separation.

N-terminal Tagging and Sequencing

• Sanger reagent (DNFB): Tags the N-terminal amino acid for identification.

Peptide and Protein Primary Structure

Sequence Notation and Determination

• Standard orientation: Sequences are written from the amino (N) terminus to the carboxy (C) terminus.

• Residue: An amino acid unit within a peptide or protein.

Edman Degradation

• PITC (phenylisothiocyanate): Reacts with the N-terminal amino acid.

• PTC (phenylthiocarbamoyl) derivative: Intermediate in the reaction.

• PTH (phenylthiohydantoin) derivative: Final product, which can be identified to determine the N-terminal residue.

• Endopeptidases: Enzymes like trypsin (cleaves after K, R) and chymotrypsin (cleaves after F, Y, W) are used to generate overlapping fragments for sequence determination.

• Disulfide bridges: Can be reduced and blocked to prevent re-oxidation, aiding in sequence analysis.

Protein Structure: Secondary, Tertiary, and Quaternary Levels

Ramachandran Plot

• Displays the allowed conformations of peptide bonds in a polypeptide chain, based on phi (φ) and psi (ψ) angles.

Peptide Bond Properties

• Amide functional group: Planar and rigid due to partial double bond character.

Levels of Protein Structure

• Primary (1°): Linear sequence of amino acids, including disulfide bridges.

• Secondary (2°): Local folding patterns stabilized by hydrogen bonds.

  • α-helix: Right-handed, 3.6 residues per turn, stabilized by H-bonds within the helix.

  • β-sheet: Parallel or antiparallel, stabilized by H-bonds between strands.

  • Turns/bends: Often contain Pro and Gly.

• Tertiary (3°): Overall 3D shape stabilized by various interactions:

  • Salt bridges (ion-ion interactions)

  • Hydrogen bonds

  • Dipole-dipole interactions

  • London dispersion forces (induced dipole-induced dipole)

  • Hydrophobic effect

• Quaternary (4°): Association of multiple polypeptide chains (e.g., hemoglobin).

Inter- and Intra-molecular Forces

• Hydrogen bonding: Involves donors (N-H, O-H) and acceptors (O, N); critical for protein folding and solubility.

• Dipole-dipole and ion-ion interactions: Contribute to protein stability and function.

• London dispersion forces: Weak, but significant in large molecules.

Cofactors and Prosthetic Groups

• Cofactor: Non-protein molecule or ion required for protein function (e.g., metal ions, vitamins).

• Prosthetic group: Tightly bound cofactor (e.g., heme in hemoglobin).

Fibrous Proteins

• α-keratin: Two α-helices intertwined, stabilized by disulfide bridges (hair, nails, horn).

• Silk/spider silk: Composed of β-sheets.

• Collagen: Triple helical structure, rich in Gly and Pro; provides tensile strength.

Hemoglobin: Structure and Function

Quaternary Structure and Allosteric Forms

• Hemoglobin (Hb): Tetramer with 2 α and 2 β subunits, forming two α/β dimers.

• T (tense) form: Deoxyhemoglobin, lower O2 affinity.

• R (relaxed) form: Oxyhemoglobin, higher O2 affinity.

• Transition between T and R forms is central to oxygen transport.

Heme Group and Oxygen Binding

• Heme: Prosthetic group that coordinates Fe2+ (ferrous iron); only Fe2+ binds O2.

• O2 binding triggers conformational changes in Hb (the "triggering mechanism").

Oxygen Binding Curve and Regulation

• O2 binding curve: Sigmoidal (S-shaped), indicating cooperative binding.

• pH (Bohr effect): Lower pH (higher [H+]) decreases O2 affinity, promoting O2 release.

• 2,3-BPG: Binds to Hb, stabilizes T form, decreases O2 affinity.

• CO2: Increases H+ (lowers pH), promotes O2 release; also binds to terminal amino groups.

Ligand Binding Equations

• Fractional saturation () of hemoglobin with O2 can be described by the Hill equation:

• Where is the Hill coefficient (degree of cooperativity), is ligand concentration, and is the dissociation constant.

Key Effects

• Bohr effect: pH sensitivity of O2 binding; lower pH favors T form and O2 release.

• Hill effect: Describes cooperativity in O2 binding; binding of one O2 increases affinity for others.

Summary Table: Protein Separation Techniques

Example: Hemoglobin Oxygen Binding

• At high pO2 (lungs), Hb binds O2 (R form favored).

• At low pO2 (tissues), Hb releases O2 (T form favored), aided by lower pH and higher CO2.

Additional info: The study of protein structure and function is foundational for understanding enzyme catalysis, metabolic regulation, and molecular medicine.