Medical School · Year 1 · Foundations · includes a quiz and discussion video

Lecture 3: Biochemistry - Amino Acids and Proteins

Unit 1.1: Foundations of Medicine & Medical Sciences

Learning Objectives

By the end of this lecture, students will be able to:

1. Classify the 20 standard amino acids by their side chain properties (nonpolar, polar, acidic, basic)
2. Describe the four levels of protein structure (primary, secondary, tertiary, quaternary) and the forces that stabilize each level
3. Explain the mechanisms of protein folding and the role of molecular chaperones
4. Identify diseases caused by protein misfolding and aggregation
5. Describe methods used to study protein structure and function
6. Apply knowledge of amino acid properties to predict protein behavior and drug interactions

Lecture Content

I. Introduction to Amino Acids

Amino acids serve as the fundamental building blocks of proteins, and understanding their structure is essential for comprehending protein behavior in biological systems. Every amino acid shares a common core architecture: a central alpha carbon that forms the hub of the molecule, connected to four different groups. These groups include an amino group (−NH₂) that gives the molecule its basic character, a carboxyl group (−COOH) that provides acidic properties, a hydrogen atom, and the variable R group or side chain that distinguishes one amino acid from another.

This tetrahedral arrangement of four different substituents around the alpha carbon creates a chiral center in all amino acids except glycine, whose R group is simply a hydrogen atom. Biological systems exclusively use the L-isomer configuration of amino acids, a selectivity that reflects the stereospecific nature of enzyme active sites and the evolutionary history of life on Earth.

When amino acids join together to form proteins, they do so through the peptide bond—a covalent linkage formed by a condensation reaction between the amino group of one amino acid and the carboxyl group of another, releasing a water molecule in the process. The peptide bond possesses unique characteristics that profoundly influence protein structure. Because resonance distributes electrons between the carbonyl oxygen and the nitrogen, the peptide bond has partial double-bond character, making it planar and rigid. This rigidity constrains the backbone of the protein, while the bonds on either side of the alpha carbon—characterized by the phi (φ) and psi (ψ) angles—retain rotational freedom. The trans configuration of the peptide bond predominates because it minimizes steric clashes between adjacent side chains.

<image>Panel A: Two separate amino acids with amino groups (blue), carboxyl groups (red), and R groups (green) positioned for reaction. Panel B: Condensation reaction with water molecule release highlighted in light blue circle. Panel C: Resulting dipeptide with peptide bond in yellow and inset showing planar C-N bond with electron resonance. Panel D: Newman projection illustrating phi and psi dihedral angles with Ramachandran plot showing allowed conformations.</image>

II. Classification of Amino Acids

The twenty standard amino acids encoded by the genetic code are classified according to the chemical properties of their side chains, a classification that directly predicts their behavior within proteins and their contributions to protein structure and function.

Nonpolar (Hydrophobic) Amino Acids

The nonpolar amino acids share the tendency to avoid water and cluster together in the interior of globular proteins, driven by the hydrophobic effect. Glycine stands apart as the smallest amino acid, with only a hydrogen atom as its side chain, which eliminates chirality and provides exceptional conformational flexibility—glycine often appears in tight turns where other residues cannot fit. Alanine carries a simple methyl group, making it mildly hydrophobic. The branched-chain amino acids—valine, leucine, and isoleucine—feature aliphatic side chains that branch at different positions and are critical for proper nutrition (all three are essential amino acids).

Proline is unique among the twenty amino acids because its side chain cyclizes back to bond with the backbone nitrogen, creating a rigid ring structure that disrupts alpha helices and introduces kinks in the polypeptide chain. Methionine contains a sulfur atom within its side chain and plays a special role as the initiator of protein synthesis (encoded by the start codon AUG). The aromatic amino acids phenylalanine and tryptophan possess ring structures that absorb ultraviolet light—tryptophan, with its bicyclic indole ring, is the largest of the standard amino acids and absorbs most strongly at 280 nm, a property exploited in protein quantification.

Polar Uncharged Amino Acids

The polar uncharged amino acids can form hydrogen bonds with water and with other polar groups, often residing on protein surfaces. Serine and threonine both carry hydroxyl groups that serve as phosphorylation sites—the addition and removal of phosphate groups at these residues represents one of the most important mechanisms of cell signaling and enzyme regulation. Cysteine contains a thiol (−SH) group capable of forming covalent disulfide bonds with other cysteine residues, creating cross-links that stabilize protein structure, particularly in secreted proteins exposed to oxidizing extracellular environments.

Asparagine and glutamine carry amide groups at the ends of their side chains and participate in hydrogen bonding. Asparagine residues in the sequence Asn-X-Ser/Thr (where X is any amino acid except proline) often serve as glycosylation sites for the attachment of carbohydrate chains. Glutamine additionally functions in nitrogen transport between tissues. Tyrosine combines aromatic and hydroxyl character—its phenolic ring can be phosphorylated by tyrosine kinases, a modification central to growth factor signaling and oncogenesis.

Acidic and Basic Amino Acids

The charged amino acids carry ionic groups at physiological pH and frequently appear on protein surfaces where they interact with water, form salt bridges, or participate in catalysis. Aspartate and glutamate carry carboxyl groups that are negatively charged at pH 7, with pKa values around 4. These residues often participate in enzyme active sites, coordinating metal ions or acting as proton donors and acceptors.

The basic amino acids—lysine, arginine, and histidine—carry positive charges. Lysine's amino group (pKa ~10.5) and arginine's guanidinium group (pKa ~12.5) remain protonated under essentially all physiological conditions. Histidine occupies a special position with a pKa near 6, meaning its imidazole ring can accept or donate protons at physiological pH—this makes histidine invaluable in enzyme catalysis and as a physiological buffer.

<image>Panel A: Nine nonpolar amino acids (Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, Trp) with gray background showing chemical structures and R groups. Panel B: Six polar uncharged amino acids (Ser, Thr, Cys, Asn, Gln, Tyr) with blue background and structures. Panel C: Two acidic amino acids (Asp, Glu) with red background showing carboxyl side chains. Panel D: Three basic amino acids (Lys, Arg, His) with purple background showing abbreviations and pKa values.</image>

III. Essential vs. Non-Essential Amino Acids

The distinction between essential and non-essential amino acids reflects the metabolic capabilities of human cells. Essential amino acids cannot be synthesized by the body and must be obtained from dietary protein sources. The mnemonic "PVT TIM HALL" helps recall the nine essential amino acids: Phenylalanine, Valine, Tryptophan, Threonine, Isoleucine, Methionine, Histidine, Arginine (conditionally essential), Leucine, and Lysine.

Non-essential amino acids can be synthesized from metabolic intermediates or from other amino acids through transamination and other reactions. This group includes alanine, asparagine, aspartate, glutamate, glutamine, glycine, proline, serine, and tyrosine (which can be made from phenylalanine).

Several amino acids occupy a middle ground as conditionally essential—they can normally be synthesized but become dietary requirements during periods of illness, physiological stress, rapid growth, or when precursors are limiting. This category includes arginine (essential during childhood and wound healing), cysteine (synthesized from methionine), glutamine (demand increases dramatically during critical illness), tyrosine (requires adequate phenylalanine), and proline.

IV. Protein Structure Hierarchy

The three-dimensional architecture of proteins is described in four hierarchical levels, each building upon the previous one and stabilized by specific molecular forces.

Primary Structure

The primary structure refers to the linear sequence of amino acids in a polypeptide chain, written by convention from the N-terminus (free amino group) to the C-terminus (free carboxyl group). This sequence is encoded in DNA and represents the fundamental information that determines all higher levels of structure. Even single amino acid substitutions can have profound consequences—the substitution of valine for glutamate at position 6 of the β-globin chain causes sickle cell disease.

Secondary Structure

Secondary structure describes the local folding patterns of the polypeptide backbone, stabilized primarily by hydrogen bonds between backbone carbonyl oxygen and amide hydrogen atoms.

The alpha helix represents one of the most common secondary structures, forming a right-handed coil with 3.6 amino acid residues per turn. Each carbonyl oxygen forms a hydrogen bond with the amide hydrogen of the residue four positions ahead in the sequence (the i to i+4 pattern). Side chains project outward from the helical axis like rungs on a spiral staircase. Alpha helices are particularly stable when composed of amino acids with appropriate helix-forming propensities and are especially common in membrane-spanning protein segments, where their hydrogen-bonded backbone provides stability in the hydrophobic lipid environment.

Beta sheets consist of extended strands aligned side by side and connected by hydrogen bonds between strands. These strands can run in the same direction (parallel) or opposite directions (antiparallel), with antiparallel sheets forming more linear, stronger hydrogen bonds. The side chains alternate above and below the plane of the sheet, creating a pleated appearance.

Turns and loops connect these regular secondary structure elements, allowing the polypeptide chain to reverse direction. Beta turns, which connect antiparallel beta strands, frequently contain glycine (for its flexibility) or proline (which naturally introduces a kink).

<image>Panel A: Alpha helix ribbon diagram in blue showing 3.6 residues per turn with hydrogen bonds as dashed green lines in i to i+4 pattern. Panel B: Alpha helix cross-sectional view from above with side chains projecting outward as ball-and-stick models. Panel C: Antiparallel beta sheet with arrows in opposite directions and perpendicular hydrogen bonds showing pleated nature. Panel D: Parallel beta sheet with same-direction arrows and angled hydrogen bonds plus Ramachandran plot inset.</image>

Tertiary Structure

Tertiary structure describes the complete three-dimensional shape of a single polypeptide chain, arising from interactions between side chains that may be far apart in the primary sequence. Multiple types of molecular interactions contribute to tertiary structure stability.

Hydrophobic interactions provide the dominant driving force, as nonpolar side chains cluster in the protein interior to minimize their contact with water. This hydrophobic collapse represents the primary event in protein folding. Hydrogen bonds form between polar side chains and between side chains and the backbone. Ionic bonds (salt bridges) occur between oppositely charged residues—for example, between a lysine and a glutamate. Disulfide bonds, unique among these interactions, are covalent bonds that form between cysteine residues and provide substantial stability, particularly important in proteins secreted into oxidizing extracellular environments. Van der Waals forces, though individually weak, contribute collectively when numerous atoms pack closely together in the protein interior.

Many proteins contain distinct structural domains—independently folding units that often correspond to functional modules. A single protein may contain a DNA-binding domain, a kinase domain, and a regulatory domain, each capable of folding autonomously. Domains represent evolutionary building blocks that have been combined in different ways to generate the diversity of proteins.

<image>Panel A: Globular protein ribbon diagram with alpha helices in red, beta sheets in yellow, and gray loops showing overall tertiary structure. Panel B: Hydrophobic core with translucent sphere containing space-filling Val, Leu, Ile, Phe side chains in gray. Panel C: Surface features showing disulfide bond between cysteines in orange and salt bridge between lysine (blue +) and glutamate (red -). Panel D: Inset showing five interaction types with relative strengths and separate protein domains diagram with distinct colored units.</image>

Quaternary Structure

Quaternary structure applies to proteins composed of multiple polypeptide subunits, describing how these subunits arrange and interact to form functional complexes. The same forces that stabilize tertiary structure—hydrophobic interactions, hydrogen bonds, ionic bonds, and occasionally disulfide bonds—operate between subunits.

Hemoglobin provides a classic example of quaternary structure, functioning as a tetramer composed of two α-globin and two β-globin chains (α₂β₂). The association of these subunits creates new properties not present in isolated subunits, particularly the cooperative oxygen binding that allows hemoglobin to load oxygen efficiently in the lungs and release it effectively in tissues. Collagen demonstrates a different quaternary arrangement—three polypeptide chains wind around each other to form a triple helix, creating the rope-like fibers that provide tensile strength to connective tissues. Antibodies consist of four polypeptides—two identical heavy chains and two identical light chains—arranged in a Y-shaped structure that places antigen-binding sites at the tips of the Y.

<image>Panel A: Hemoglobin tetramer ribbon format with two alpha subunits in shades of blue and two beta subunits in shades of red. Panel B: Four heme groups as flat orange-brown discs in binding pockets with iron atoms as red spheres at centers. Panel C: Central cavity between subunits with highlighted interface regions where subunits contact. Panel D: Insets showing single globin subunit with heme group and O2 molecule approaching iron center with scale bar.</image>

V. Protein Folding

One of the most remarkable aspects of protein biology is that the linear amino acid sequence contains all the information needed to specify the three-dimensional structure. Understanding how proteins fold has been called "the protein folding problem."

The Protein Folding Problem and Levinthal's Paradox

If a protein were to explore all possible conformations randomly, it would never fold on any reasonable timescale—this is Levinthal's paradox. A 100-residue protein has approximately 10⁸⁰ possible conformations. Even if each conformation could be sampled in a picosecond, exhaustive sampling would require longer than the age of the universe. Yet proteins fold within milliseconds to seconds. This observation tells us that folding cannot be a random search but must follow defined pathways through a funnel-shaped energy landscape, with the native state representing the thermodynamic free energy minimum.

Thermodynamics of Folding

The thermodynamic driving force for folding is the decrease in free energy achieved in the native state compared to the unfolded ensemble. The hydrophobic effect dominates: sequestering nonpolar side chains in the protein interior releases ordered water molecules from around those hydrophobic groups, increasing entropy and driving folding forward. This entropy gain from water release compensates for the entropy loss of the polypeptide chain itself, which becomes more ordered upon folding.

Molecular Chaperones

Although proteins contain all the information needed for folding, the cellular environment presents challenges. Newly synthesized proteins emerge from ribosomes into a crowded cytoplasm where they might aggregate with other unfolded proteins before achieving their native states. Molecular chaperones are proteins that assist folding without becoming part of the final structure—they act as folding catalysts and aggregation preventers.

The heat shock proteins (HSPs) constitute major families of chaperones, named for their upregulation during cellular stress. Hsp70 chaperones recognize and bind exposed hydrophobic patches on nascent or denatured proteins, preventing premature folding or aggregation. ATP hydrolysis drives cycles of binding and release that give the substrate protein repeated opportunities to fold correctly.

The Hsp60 chaperonins (GroEL/GroES in bacteria) provide an entirely different mechanism—they create an isolated folding chamber. An unfolded protein enters the barrel-shaped GroEL complex, and the lid-like GroES caps the chamber. Inside this protected environment, the protein can fold without the risk of aggregation with other cellular proteins. ATP hydrolysis triggers conformational changes that eventually release the folded protein.

Hsp90 chaperones work with more specialized clients, particularly signaling proteins like steroid hormone receptors and kinases, holding them in states competent to respond to regulatory signals.

<image>Panel A: GroEL double-ring barrel in blue with unfolded protein (tangled red line) approaching and binding to hydrophobic cavity surfaces. Panel B: ATP binding (yellow stars) triggering conformational changes and GroES cap (green dome) attaching to enlarge folding chamber. Panel C: Protein folding inside protected chamber shown as red line adopting compact organized structure. Panel D: ATP hydrolysis triggering GroES release and folded protein ejection with cis/trans ring alternation diagram.</image>

VI. Protein Misfolding and Disease

When proteins fail to achieve or maintain their native conformations, the consequences can be severe. Misfolded proteins may lose their normal functions, gain toxic properties, or aggregate into insoluble deposits that damage tissues.

Amyloid Diseases

A diverse group of diseases share a common pathological feature: the deposition of proteins in an abnormal fibrillar form called amyloid. Despite involving different proteins, all amyloid fibrils share a characteristic cross-β structure in which β-strands run perpendicular to the fiber axis, creating a highly stable architecture resistant to proteolysis.

Alzheimer's disease involves the aggregation of two different proteins: the Aβ peptide, derived from proteolytic processing of amyloid precursor protein, forms extracellular plaques, while the microtubule-associated protein tau aggregates into intracellular neurofibrillary tangles. Parkinson's disease features aggregation of α-synuclein into inclusions called Lewy bodies within dopaminergic neurons of the substantia nigra. In type 2 diabetes, the peptide hormone amylin (islet amyloid polypeptide, IAPP), normally co-secreted with insulin, forms amyloid deposits in pancreatic islets that contribute to β-cell death. Systemic amyloidoses can involve various proteins depositing in multiple organs, with light-chain amyloidosis (from immunoglobulin light chains) and transthyretin amyloidosis being among the more common forms.

Prion Diseases

The transmissible spongiform encephalopathies represent a unique category of protein misfolding disease. The normal cellular prion protein (PrP^C) is a membrane-anchored protein of unknown function that is predominantly α-helical in structure. In prion diseases, this protein converts to an abnormal form (PrP^Sc) that is rich in β-sheet structure and aggregates into amyloid. The remarkable feature of prions is that PrP^Sc acts as a template, inducing normal PrP^C molecules to adopt the pathogenic conformation—a self-propagating chain reaction that allows these diseases to be infectious even though no nucleic acid is involved.

Human prion diseases include Creutzfeldt-Jakob disease (which can arise sporadically, be inherited, or be acquired through contaminated tissue), kuru (historically transmitted through ritual cannibalism), and variant CJD (linked to bovine spongiform encephalopathy, or "mad cow disease").

<image>Panel A: Normal PrP^C ribbon diagram with alpha-helical structure (green spirals), unstructured N-terminus, and GPI membrane anchor. Panel B: Misfolded PrP^Sc with extensive beta-sheet structure (red arrows) in compact aggregation-prone shape. Panel C: Template-directed conversion showing PrP^Sc contacting PrP^C, conformational conversion at interface, and propagation. Panel D: Amyloid fibril formation with stacked PrP^Sc molecules in cross-beta structure and brain silhouette with spongy lesions.</image>

Endoplasmic Reticulum Stress and the Unfolded Protein Response

The endoplasmic reticulum (ER) serves as the folding compartment for secreted and membrane proteins, containing abundant chaperones and an oxidizing environment conducive to disulfide bond formation. When misfolded proteins accumulate in the ER—due to mutations, cellular stress, or overwhelming protein load—the unfolded protein response (UPR) is activated. This signaling pathway initially attempts to restore homeostasis by reducing protein synthesis, increasing chaperone production, and enhancing degradation of misfolded proteins. If these measures fail to resolve the stress, the UPR triggers apoptosis.

Alpha-1 antitrypsin deficiency illustrates ER stress in disease. The Z variant of this serine protease inhibitor misfolds and accumulates in the ER of hepatocytes rather than being secreted. This leads to ER stress and liver damage, while the deficiency of circulating α1-antitrypsin permits uncontrolled elastase activity in the lungs, causing emphysema.

VII. Methods to Study Proteins

Our understanding of protein structure and function has advanced through increasingly sophisticated experimental techniques.

Structural Methods

X-ray crystallography has determined more protein structures than any other method. The technique requires growing well-ordered protein crystals, which are then exposed to X-ray beams. The resulting diffraction pattern is mathematically transformed to generate an electron density map from which the atomic structure is built. Crystallography can achieve atomic resolution (better than 2 Å) and has revealed the structures of proteins ranging from small enzymes to the ribosome. However, it provides a static snapshot of what is inherently a dynamic molecule.

Nuclear magnetic resonance (NMR) spectroscopy studies proteins in solution, providing information about dynamics and conformational flexibility as well as structure. NMR is limited to relatively small proteins (generally under 40 kDa) because larger molecules produce overlapping signals that become impossible to interpret, but it offers unique insights into protein motions on timescales from picoseconds to seconds.

Cryo-electron microscopy (cryo-EM) has undergone a resolution revolution in recent years. Samples are flash-frozen in vitreous ice, preserving them in their native hydrated state without the need for crystallization. Thousands of images of individual particles in different orientations are computationally combined to reconstruct three-dimensional structures. Cryo-EM excels at visualizing large complexes and membrane proteins that resist crystallization, with recent advances pushing resolution toward the atomic level.

Analytical Methods

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins by molecular weight after denaturation with the detergent SDS, which imparts uniform negative charge. Western blotting (immunoblotting) combines SDS-PAGE with antibody-based detection to identify specific proteins. Mass spectrometry provides precise molecular weight measurements and can sequence proteins by fragmenting them and analyzing the masses of the resulting peptides. Circular dichroism spectroscopy measures differential absorption of left- and right-handed circularly polarized light, providing information about secondary structure content.

<image>Panel A: X-ray crystallography showing protein crystal in loop mount, X-ray beam producing diffraction pattern on detector, and electron density map with fitted model. Panel B: NMR spectroscopy with sample in tube entering superconducting magnet, spectrum with chemical shift peaks, and conformational ensemble bundle. Panel C: Cryo-EM showing sample grid with vitreous ice, electron microscope column, and raw micrograph with fuzzy particle spots. Panel D: Cryo-EM 2D class averages and final 3D reconstruction surface with fitted atomic model and technique comparison icons.</image>

VIII. Clinical Applications

Knowledge of protein biochemistry translates directly into medical practice through therapeutic proteins, diagnostic applications, and emerging treatments targeting protein misfolding.

Therapeutic Proteins

Recombinant DNA technology has enabled production of human proteins for therapeutic use. Insulin was the first recombinant protein approved for clinical use (1982), revolutionizing diabetes treatment by providing unlimited supplies of human insulin rather than animal-derived preparations. Growth hormone treats pituitary deficiency, and erythropoietin stimulates red blood cell production in chronic kidney disease. Monoclonal antibodies represent the fastest-growing class of therapeutics, with applications spanning oncology (targeting tumor antigens), autoimmune disease (neutralizing inflammatory cytokines), and infectious disease. Enzyme replacement therapy provides functional enzymes to patients with lysosomal storage disorders such as Gaucher disease and Fabry disease.

Protein-Based Diagnostics

Many diagnostic tests rely on detecting or measuring specific proteins. Enzyme-linked immunosorbent assay (ELISA) uses antibodies to detect protein analytes with high sensitivity and specificity. Cardiac troponins serve as biomarkers for myocardial infarction—their release from damaged cardiac muscle into the bloodstream provides both diagnostic and prognostic information. Prostate-specific antigen (PSA) screening, while controversial, exemplifies the use of protein biomarkers in cancer detection. Autoantibody testing helps diagnose autoimmune diseases and paraneoplastic syndromes.

Targeting Protein Misfolding

Pharmacological chaperones are small molecules that bind to and stabilize mutant proteins, promoting proper folding and trafficking. Tafamidis, for example, stabilizes transthyretin tetramers and slows progression of transthyretin amyloidosis. Antisense oligonucleotides can reduce expression of disease-causing proteins—patisiran and inotersen reduce transthyretin production in hereditary transthyretin amyloidosis. Gene therapy and CRISPR-based approaches offer the possibility of correcting the underlying genetic mutations that cause protein misfolding diseases.

Summary

Amino acids are the building blocks of proteins, classified by their side chain properties into nonpolar, polar uncharged, acidic, and basic categories. These properties determine where amino acids reside within protein structures and how they contribute to protein function. Protein structure is organized hierarchically: primary structure (amino acid sequence) determines secondary structure (local folding patterns like α-helices and β-sheets), which combines to form tertiary structure (the complete three-dimensional shape), and multiple subunits may associate to create quaternary structure. Proper protein folding is essential for function and is assisted by molecular chaperones including the Hsp70 and Hsp60 families. When proteins misfold, they can aggregate into amyloid fibrils, causing diseases ranging from Alzheimer's to systemic amyloidoses. Prion diseases represent a special case where misfolded protein becomes infectious by templating the conversion of normal protein. Multiple techniques including X-ray crystallography, NMR, and cryo-EM allow us to determine protein structures, while therapeutic proteins and treatments targeting misfolding represent important clinical applications.

Key Terms

This content is subject to the MIT License. © 2024–2026 Hibbert School of Medicine.

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