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 all proteins (except some bacterial cell walls and antibiotics).

• In Fischer projections, the L isomer has the amino group on the left.

Classification by R Groups

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

• Non-polar (hydrophobic): e.g., Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Proline

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

• Polar, uncharged: e.g., Serine, Threonine, Cysteine, Asparagine, Glutamine

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

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

Ionization, pKa, and Buffering

• Amino acids have ionizable groups (amino, carboxyl, and sometimes R groups) with characteristic pKa values.

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

• Titration curves can be used to determine pKa values and pI, and to predict the predominant species at a given pH.

• The Henderson-Hasselbalch equation is used to relate pH, pKa, and the ratio of protonated to deprotonated forms:

• This equation is useful for buffer preparation and calculating concentrations of species at a given pH.

Solubility and pKa Values

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

• The pKa values for the amino and carboxyl termini differ between free amino acids and those in peptides.

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 points (pI).

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

N-terminal Tagging and Sequencing

• Sanger reagent (DNFB): reacts with N-terminal amino acids for identification.

Peptide and Protein Primary Structure

Sequence and Nomenclature

• Peptide sequences are written from the amino (N) terminus to the carboxy (C) terminus.

• Each amino acid in a peptide is called a residue.

Edman Degradation

• Edman degradation is a method for sequencing peptides by removing one residue at a time from the N-terminus.

• Key reagents and intermediates: PITC (phenylisothiocyanate), PTC (phenylthiocarbamoyl derivative), PTH (phenylthiohydantoin derivative).

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

• Disulfide bonds can be reduced and blocked to prevent re-oxidation, which is important for determining primary structure.

Protein Structure: Secondary, Tertiary, and Quaternary Levels

Ramachandran Plot

• A Ramachandran plot displays the allowed conformations of peptide bonds (phi and psi angles) in a polypeptide chain.

Peptide Bond Properties

• The peptide bond is an amide linkage, planar, and rigid due to partial double-bond character.

Levels of Protein Structure

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

• 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 structure stabilized by:

  • 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: Occurs between donors (N-H, O-H) and acceptors (O, N); important for solubility and structure.

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

• 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 and spider silk: Composed of β-sheets.

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

Hemoglobin: Structure and Function

Quaternary Structure and Allostery

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

• Exists in two forms:

  • T (tense, deoxy) form: Lower affinity for O2

  • R (relaxed, oxy) form: Higher affinity for O2

• Transition between T and R forms is central to hemoglobin's function.

Heme Group and Oxygen Binding

• Heme group: Contains Fe2+ (ferrous iron); only Fe2+ binds O2 (Fe3+ is inactive).

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

Oxygen Binding Curve and Regulation

• Hemoglobin's O2 binding curve is sigmoidal (S-shaped), indicating cooperativity.

• Curve shifts in response to:

  • pH (Bohr effect): Lower pH (higher [H+]) decreases O2 affinity (favors T form).

  • 2,3-BPG: Binds to deoxy-Hb, stabilizing the T form and promoting O2 release.

  • CO2: Increases H+ and Cl– concentration, favoring T form and O2 delivery to tissues.

• Hill coefficient: Quantifies cooperativity in ligand binding.

Ligand Binding Equations

• The fraction of occupied binding sites (Y) is given by:

• For hemoglobin, the equation is modified to account for cooperativity (Hill equation):

• Where n is the Hill coefficient.

Summary Table: Hemoglobin Regulation

Bohr and Hill Effects

• Bohr effect: pH sensitivity of O2 binding; lower pH promotes O2 release.

• Hill effect: Describes cooperative binding of O2 to hemoglobin.

Protein-Ligand Interactions

• Ligand binding is described by equilibrium equations and is influenced by ligand concentration and protein structure.

Example:

At high altitude, increased 2,3-BPG levels in red blood cells promote O2 release to tissues by stabilizing the T form of hemoglobin.

Additional info: The above notes expand on the study guide by providing definitions, equations, and examples for each topic, ensuring a self-contained and comprehensive review for exam preparation.