Biochemistry Study Guide: Foundations, Chemical Principles, Genetics, and Protein Analysis (Lectures 1–7)

Foundations of Biochemistry

Molecular Foundations of Life

Biochemistry studies the molecular mechanisms of life, focusing on how nucleic acids, proteins, lipids, and carbohydrates interact and power cells. Life is constrained by chemistry (reaction rates, thermodynamics/kinetics) and physics (energy, forces).

• Cells: Fundamental Unit – Prokaryotes (no nucleus) vs. Eukaryotes (nucleus). Domains: Bacteria, Archaea, Eukarya.

• Energy & Resources – Phototrophs harvest light; Chemotrophs oxidize chemicals. Autotrophs fix CO2; Heterotrophs require organic carbon.

• Organelles (Eukaryotes) – Mitochondria, ER, Golgi, lysosomes, peroxisomes, chloroplasts (plants), vacuoles.

• Cytoskeleton – Actin, microtubules, intermediate filaments organize the cytoplasm; highly dynamic.

Example: Place humans in the energy/carbon classification (chemoheterotrophs).

Chemical Foundations & Stereochemistry

Functional Groups and Bonding

Chemical structure determines biological function. Functional groups (e.g., alcohols, aldehydes, ketones, carboxylic acids, amines, amides, thiols, phosphates) define reactivity and interactions.

• Macromolecules – Proteins (amino acids), nucleic acids (nucleotides), polysaccharides (sugars), lipids (fatty acids).

• Configuration vs. Conformation – Configuration: fixed spatial arrangement (e.g., R/S, D/L); Conformation: rotatable arrangement without breaking covalent bonds.

• Thermo Frame – Cells are open systems in a dynamic steady state, not equilibrium; maintaining order requires energy.

Example: Recognize a carboxyl group and its role in amino acids.

Genetic Foundations

DNA Structure and Replication

Genetic information is encoded in the linear sequence of deoxyribonucleotides in DNA, which stores instructions and templates for replication.

• Continuity & Fidelity – Complementary base pairing and hydrogen bonding enable accurate replication and repair.

• Scale Example – E. coli genome: a single circular DNA molecule with ~4.6 × 106 base pairs.

• Sequence → Structure → Function – DNA sequence determines protein sequence, which determines native 3D conformation and function.

Example: Explain why hydrogen bonds are essential for replication fidelity.

Water, pH, and Buffers

Properties of Water and pH Regulation

Water is special due to extensive hydrogen bonding, high melting/boiling points, and excellent solvent properties for polar/charged solutes. The hydrophobic effect drives micelle formation, protein folding, and membrane assembly.

• pH & Ionization – Water, pH, and pKa concepts; proton hopping.

• Henderson-Hasselbalch Equation – Relates pH, pKa, and acid/base titration curves.

• Buffers – Most effective when pH ≈ pKa; biological systems use phosphate and bicarbonate buffers. Histidine side chains buffer proteins near physiological pH.

Example: Identify the best buffer for pH 7.2 from a list of candidate pairs.

Amino Acids, Peptides, Proteins

Structure and Properties of Amino Acids

Amino acids are the building blocks of proteins. Most amino acids in proteins are L-stereoisomers (except glycine, which is achiral). Each has a carboxyl group, aminoR group, and a central carbon.

• Acid-base Behavior – Zwitterions at neutral pH; titration curves reveal pKa values for carboxyl, amino, and side chains.

• Peptides – Formed by condensation (amide bond formation); hydrolysis reverses the process.

• Residues – Peptide bond links amino acids; residue mass = MW/110.

Example: Rank side chains by likely location (core vs. surface) in a soluble protein.

Protein Expression & Purification

Methods and Principles

Proteins are expressed in hosts (E. coli, yeast, insect, mammalian cells) and purified using various methods.

• Expression Choices – Host selection affects folding, PTMs, yield.

• Separation Principles – Purify by size, charge, binding affinity, solubility. His-tag/Ni-NTA affinity purification is common for tagged proteins.

• Electrophoresis Setup – Use polyacrylamide gels, SDS, and buffers to resolve proteins by size.

Example: Given pI = 7.2 and pH 6.0 vs. 8.5, which ion-exchange method purifies your protein?

Working with Proteins: Chromatography, Gels, MS

Analytical and Preparative Techniques

Proteins are analyzed and purified using chromatography, gel electrophoresis, and mass spectrometry.

• Chromatography Types – Ion-exchange: separates by net charge (controlled by pH and salt). Size-exclusion: separates by size. Affinity: specific binding (e.g., His-tag/Ni-NTA).

• SDS-PAGE – Denatures and separates proteins by mass; migration ∝ log(MW).

• Proteolysis & Sequencing – Proteases (trypsin, chymotrypsin, V8) and mass spectrometry (LC/MS/MS) identify protein sequence and modifications.

Example: Which column resolves two proteins with same MW and pI but different ligand binding?

Formulas & Quick Facts

Essential Equations and Data

• Henderson-Hasselbalch Equation: $\mathrm{pH} = \mathrm{p}K_a + \log\left(\frac{[\mathrm{A}^-]}{[\mathrm{HA}]}\right)$

• Amino Acid Residue Estimate: $\text{Residues} = \frac{\text{MW}}{110}$

• pI (no ionizable side chain): $\text{pI} = \frac{\text{p}K_{\text{COOH}} + \text{p}K_{\text{NH}_3}}{2}$

• Dynamic Steady State: Living cells maintain concentrations far from equilibrium using constant energy input.

Practice Prompts

Application and Review

• Buffers: Choose the best buffer pair for pH 7.0 ± 0.05; compute buffer pH after adding strong base.

• Amino Acids: Classify 10 residues; order those absorbing at 280 nm; estimate peptide pI.

• Chromatography: Plan a 3-step purification for a His-tagged enzyme with pI 6.0; justify order.

• SDS-PAGE: Predict lane pattern after DTT reduction of a disulfide-linked dimer.

• Genetics: Explain how base pairing enables high-fidelity replication in 3 sentences.

How to Use This Guide Efficiently

• Spaced recall: Review, answer prompts, then verify.

• Make mini-cards: For functional groups, amino acid classes, chromatography rules of thumb.

• Practice: Use both conceptual and quantitative questions for exam prep.