Fundamental Concepts in Biochemistry: Amino Acids, Protein Structure, and Purification Techniques

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Amino acids are the building blocks of proteins, each with distinct side chains that determine their chemical properties and biological roles.

20 Common Amino Acids: Each has a unique side chain (R group) affecting polarity, charge, and aromaticity.
Stereochemistry: Most amino acids are chiral, existing as L- and D- enantiomers; proteins are composed of L-amino acids.
Classification: Amino acids can be categorized as non-polar, polar, aromatic, acidic, or basic.
One and Three Letter Codes: Each amino acid is represented by a standard one-letter and three-letter code (e.g., Gly/G for glycine).

Example: Glycine (Gly, G) is non-polar and achiral; Glutamic acid (Glu, E) is acidic and negatively charged at physiological pH.

Amino acids contain ionizable groups (amino, carboxyl, and sometimes side chains) whose charge depends on pH.

pKa Values: Each ionizable group has a characteristic pKa, determining its protonation state at a given pH.
Isoelectric Point (pI): The pH at which the amino acid has no net charge.

Equation:

Proteins are polymers of amino acids linked by peptide bonds, forming a backbone known as the main chain.

Peptide Bond: A covalent bond between the carboxyl group of one amino acid and the amino group of another, with partial double-bond character restricting rotation.
Main Chain: The repeating backbone structure of a peptide, excluding side chains.
Peptide Group: Six atoms in a plane: Cα, C, O, N, H (from the peptide bond).

Example: The sequence Ala-Gly-Ser forms two peptide bonds and a tripeptide main chain.

Polypeptides have distinct ends:

At physiological pH, amino acids exist as zwitterions, carrying both positive (amino) and negative (carboxyl) charges.

Genes encode proteins via sequences of nucleotides, which are transcribed and translated.

Coding Strand: The DNA strand whose sequence matches the mRNA (except T/U).
Non-coding Strand: The template for RNA synthesis.
Open Reading Frame (ORF): A stretch of DNA with a start codon and no stop codon until the end, potentially encoding a protein.

Conservative Mutation: Alters the amino acid to one with similar properties (size, charge, polarity).
Non-conservative Mutation: Changes the amino acid to one with different properties, potentially affecting protein function.

This technique separates proteins based on size by passing them through a column filled with porous beads.

Principle: Larger proteins elute first as they are excluded from the pores; smaller proteins elute later.

Proteins are separated based on charge by passing them through a column with charged beads.

Cation Exchange: Binds positively charged proteins.
Anion Exchange: Binds negatively charged proteins.
Elution: Proteins are eluted by increasing salt concentration or changing pH.

SDS-PAGE is an analytical technique for separating proteins by size.

SDS: Denatures proteins and imparts a uniform negative charge.
Polyacrylamide Gel: Acts as a molecular sieve; smaller proteins migrate faster.

Affinity chromatography separates proteins based on specific binding interactions.

Principle: Proteins with high affinity for a ligand immobilized on the column are retained, while others elute.

Term	Definition
Chiral	A carbon center that can exist as right- or left-handed mirror images (enantiomers).
Fischer Projection	Depiction of an organic molecule (carbon center) in which all bonds are represented as solid lines; horizontal bonds are viewed as projecting toward the viewer, vertical bonds away.
Zwitterion	Electrically neutral form of an amino acid with both positive and negative charges.
Peptide Group	Six atoms in a plane that include the N and C involved in the peptide bond.
Conservative Mutation	Mutation that changes the chemical nature of the side chain to a similar type.
Non-conservative Mutation	Mutation that changes the chemical nature of the side chain to a different type.
Coding Strand	The DNA strand whose sequence matches the RNA transcript.
Non-coding Strand	The DNA strand that serves as the template for RNA synthesis.
Open Reading Frame (ORF)	A section of DNA that contains a continuous sequence between a potential start codon and stop codon.

Understanding the peptide bond's planarity and restricted rotation is crucial for protein secondary structure.
Protein purification techniques are essential for isolating proteins for biochemical analysis and research.
Mutations in coding regions can affect protein structure and function, with conservative mutations generally less disruptive than non-conservative ones.