Chapter 1: Amino Acids, Peptides, and Proteins

Every amino acid in your body is built on the same four-part chassis. Like a car - wheels, engine, frame, seats - the parts never change. What changes is the paint job. That paint job is the side chain, and it decides whether the amino acid is greasy, charged, acidic, basic, bulky, or tiny.

If you understand one amino acid, you understand the structural skeleton of all 20.

The Four Groups Around the Central Carbon

Every standard amino acid has a central carbon (the alpha carbon, Cα) with four things attached:

1. An amino group (-NH2 or -NH3+ at physiological pH)
2. A carboxyl group (-COOH or -COO- at physiological pH)
3. A hydrogen atom
4. An R group (the side chain - this is what varies)

The name “amino acid” comes straight from the structure: an “amino” group and a carboxylic “acid” group on the same carbon.

The Alpha Carbon is Chiral (Except in Glycine)

Because the alpha carbon has four different groups attached in 19 of the 20 standard amino acids, it is a chiral center. That means it has two non-superimposable mirror images: L and D forms.

Biological proteins use L-amino acids almost exclusively. Your ribosomes only build with the L-form. D-amino acids do exist (bacterial cell walls use some, and a few show up in venoms and signaling peptides) but they are passage-peek rare on the MCAT.

Glycine is the exception. Its R group is just a hydrogen atom, so the alpha carbon has two identical hydrogens. No chirality. Glycine is achiral.

Why the Structure Matters

The amino and carboxyl groups on the central carbon are what allow amino acids to link together into chains. The carboxyl of one amino acid reacts with the amino of the next, forming a peptide bond and releasing water. That reaction happens millions of times per second on a ribosome. We cover the mechanism in peptide bond formation.

The R group is what makes each amino acid unique. Some R groups are just plain alkyl chains (greasy). Some have polar OH groups. Some have carboxylic acids (negatively charged) or amines (positively charged) built in. Some have aromatic rings. A tiny few do special tricks (like cysteine forming disulfide bridges). We sort all 20 of them in the next section.

Memorizing 20 random structures is a slog. Grouping them by what their R group does is a cheat code. Once you see that histidine, lysine, and arginine all have nitrogen that grabs a proton and becomes positive, you know what they do in a protein regardless of which one the passage names.

The 20 standard amino acids split into five main buckets plus three special cases. Learn the buckets first. Individual structures come in the next section.

The Five R-Group Buckets

Nonpolar R Groups (Hydrophobic)

These have alkyl chains, sulfur atoms (in methionine), or aromatic rings with no polar groups. They are greasy. In a folded protein, they huddle in the interior away from water - the hydrophobic core. Aromatic residues also stack with each other for extra stability.

Polar Uncharged R Groups

These have heteroatoms (O, N, S) that form hydrogen bonds but do not carry a net charge at pH 7. They love water. They sit on the protein surface or in active sites where they can hydrogen bond to substrates.

• Serine, Threonine - have -OH groups
• Tyrosine - aromatic ring with -OH (classified polar or aromatic depending on the chart)
• Cysteine - has -SH (thiol)
• Asparagine (N), Glutamine (Q) - have amide side chains

Positively Charged R Groups (Basic)

Three amino acids. Their side chains carry a nitrogen lone pair that picks up a proton and becomes positively charged at pH 7.

• Lysine (K) - long chain ending in -NH3+
• Arginine (R) - guanidinium group, very basic (pKa about 12, fully protonated at pH 7)
• Histidine (H) - imidazole ring, pKa near 6, so at pH 7 it is partly protonated. This makes histidine uniquely useful in enzyme active sites that need to shuttle protons.

Negatively Charged R Groups (Acidic)

Two amino acids, both ending in “-ate” because they donate their carboxyl proton at pH 7.

• Aspartate (Asp, D) - one -CH2- linker, then -COO-
• Glutamate (Glu, E) - two -CH2- linkers, then -COO-

Their protonated forms are called aspartic acid and glutamic acid, but at physiological pH they are almost always in the deprotonated “-ate” form.

The Three Special Cases

Glycine (Gly, G). R group is just -H. Smallest amino acid. Only achiral one. Flexible - fits anywhere. Often found at tight turns in a protein.

Proline (Pro, P). Its R group loops back and bonds to the amino nitrogen, forming a rigid five-membered ring. This kink disrupts alpha helices and creates helix breakers or sharp bends.

Cysteine (Cys, C). Its -SH (thiol) can form a covalent disulfide bond (-S-S-) with another cysteine. This is the only covalent bond (besides peptide bonds) that stabilizes folded proteins.

You need to know all 20 by structure, name, three-letter code, one-letter code, and category. On test day you may see any of these formats in a passage and have to reason about it instantly. The table below is worth memorizing cold.

The Master Table

| Name | 3-letter | 1-letter | Category | Side chain feature | pKa of R group (if any) |
|------|----------|----------|----------|--------------------|-------------------------|
| Glycine | Gly | G | Special (nonpolar) | -H (achiral) | - |
| Alanine | Ala | A | Nonpolar aliphatic | -CH3 | - |
| Valine | Val | V | Nonpolar aliphatic (branched) | -CH(CH3)2 | - |
| Leucine | Leu | L | Nonpolar aliphatic (branched) | -CH2CH(CH3)2 | - |
| Isoleucine | Ile | I | Nonpolar aliphatic (branched) | -CH(CH3)CH2CH3 | - |
| Methionine | Met | M | Nonpolar, sulfur | -CH2CH2SCH3 | - |
| Proline | Pro | P | Special (nonpolar) | Cyclic, bonds back to N | - |
| Phenylalanine | Phe | F | Aromatic (nonpolar) | -CH2-C6H5 | - |
| Tryptophan | Trp | W | Aromatic (nonpolar) | Indole ring | - |
| Tyrosine | Tyr | Y | Aromatic (polar) | Phenol ring (-OH) | ~10 |
| Serine | Ser | S | Polar uncharged | -CH2OH | - |
| Threonine | Thr | T | Polar uncharged | -CH(OH)CH3 | - |
| Cysteine | Cys | C | Special (polar) | -CH2SH | ~8.3 |
| Asparagine | Asn | N | Polar uncharged | -CH2CONH2 | - |
| Glutamine | Gln | Q | Polar uncharged | -CH2CH2CONH2 | - |
| Aspartate | Asp | D | Acidic (negative) | -CH2COO- | ~3.9 |
| Glutamate | Glu | E | Acidic (negative) | -CH2CH2COO- | ~4.1 |
| Lysine | Lys | K | Basic (positive) | -(CH2)4NH3+ | ~10.5 |
| Arginine | Arg | R | Basic (positive) | Guanidinium | ~12.5 |
| Histidine | His | H | Basic (partial +) | Imidazole | ~6.0 |

The alpha-amino group of any free amino acid has a pKa around 9 to 10. The alpha-carboxyl group has a pKa around 2. Memorize these two values - they appear in every titration question.

Essential vs. Nonessential

Humans can synthesize 11 of the 20 amino acids from scratch. The other 9 must come from diet - these are the essential amino acids.

Amino Acids with Ionizable Side Chains

Seven amino acids have R groups with ionizable protons. These are the only ones whose charge state depends on pH, and they are the ones you will see on titration and isoelectric-point questions.

| Amino acid | R-group pKa | Charge below pKa | Charge above pKa |
|------------|-------------|------------------|------------------|
| Asp (D) | 3.9 | 0 | -1 |
| Glu (E) | 4.1 | 0 | -1 |
| His (H) | 6.0 | +1 | 0 |
| Cys (C) | 8.3 | 0 | -1 |
| Tyr (Y) | 10.1 | 0 | -1 |
| Lys (K) | 10.5 | +1 | 0 |
| Arg (R) | 12.5 | +1 | 0 |

How to Study This Table

Do not try to memorize everything at once. Start with categories (previous section). Then memorize the three-letter codes by writing them out with structures five times. Then add the one-letter codes. Then pKa values for the seven ionizable ones. This layered approach beats rote flashcards.

Amino acids have two ionizable groups always (the alpha amino and alpha carboxyl) plus one more if the R group is ionizable. At any given pH, some of those groups are protonated and some are not. Combining those charge states across three groups gives every amino acid its personality on a titration curve.

You will see this concept tested two ways: (1) “what is the charge at pH X” and (2) “what is the pI.” Master both and you can handle every acid-base amino acid question.

The Zwitterion

A zwitterion is a molecule with both a positive and a negative charge that cancel out to give a net charge of zero. At physiological pH (7.4), every amino acid with a nonionizable R group exists almost entirely as a zwitterion: the alpha amino group is protonated (-NH3+) and the alpha carboxyl is deprotonated (-COO-).

Titration Curve of a Simple Amino Acid (Glycine)

Glycine has two ionizable groups. Titrating from low pH (excess H+) with added base gives a curve with two plateaus and three charged species.

At very low pH: fully protonated. Both -NH3+ and -COOH. Net charge = +1.

As pH rises past pKa1 (~2.3): the -COOH loses its proton to become -COO-. Now -NH3+ and -COO-. Net charge = 0. This is the zwitterion. The flat region near pKa1 is a buffering zone.

As pH rises past pKa2 (~9.6): the -NH3+ loses its proton to become -NH2. Now -NH2 and -COO-. Net charge = -1.

Calculating the Isoelectric Point (pI)

The pI is the pH at which the amino acid carries no net charge. For an amino acid with only the two backbone ionizable groups, the pI is simply:

With an Ionizable Side Chain

If the R group is also ionizable, the amino acid has three pKa values. The pI is the average of the two pKas that flank the zwitterionic (net-zero) form.

• Acidic amino acid (Asp, Glu): pI = average of the two lowest pKas (both the side-chain carboxyl and the alpha carboxyl lose protons to produce the neutral form). The pI is low (around 3 to 4).
• Basic amino acid (Lys, Arg, His): pI = average of the two highest pKas (both amino/basic groups must still be protonated for the zwitterion). The pI is high (around 7.6 to 11).
• Neutral amino acid with an ionizable side chain (Cys, Tyr): treat it like a neutral amino acid; pI is the average of the alpha-carboxyl pKa and the alpha-amino pKa.

Worked Examples: Three Types, Three Calculations

The MCAT loves these. Work through all three types once and the pattern will feel obvious on test day.

The shortcut: acidic side chain → pI = average of the two lowest pKas; basic side chain → pI = average of the two highest pKas. This works because you always sandwich the zwitterion between the two pKas closest to it on the protonation ladder.

Charge at Any pH

Two simple rules beat every “what is the charge” question:

1. If pH is below pKa, the group is protonated.
2. If pH is above pKa, the group is deprotonated.

For an amine (-NH3+ / -NH2): protonated is +1, deprotonated is 0.
For a carboxyl (-COOH / -COO-): protonated is 0, deprotonated is -1.

Apply the rule to every ionizable group on the molecule and sum the charges.

Henderson-Hasselbalch for Amino Acids

The Henderson-Hasselbalch equation applies to each ionizable group:

Snap LEGO bricks together. That is peptide bond formation. Take the carboxyl group of one amino acid, the amino group of the next, pop off a water molecule, and you have a covalent amide bond linking the two. Do this 146 times in a row and you have the hemoglobin beta chain. Do it 1,480 times and you have a mid-size enzyme.

This is the reaction that builds every protein in your body, at a rate of about 10 to 20 amino acids per second on a ribosome.

The Mechanism

The -COOH of amino acid #1 reacts with the -NH2 of amino acid #2. The -OH of the carboxyl leaves together with one H from the amino group - that is the water molecule released.

What remains is a covalent bond from the carbonyl carbon of amino acid #1 directly to the nitrogen of amino acid #2. The functional group -C(=O)-N(H)- is an amide. A peptide bond is just a specific kind of amide bond that connects two amino acids.

Naming the Ends: N-Terminus and C-Terminus

Every polypeptide has two ends because each peptide bond uses up one -NH2 and one -COOH but leaves the amino group of the first residue and the carboxyl of the last residue free.

• The N-terminus has a free alpha amino group. This is where translation starts.
• The C-terminus has a free alpha carboxyl group. This is where translation ends.

Peptide sequences are always written N-terminus to C-terminus, left to right. “Ala-Gly-Ser” means alanine at the N-terminus, glycine in the middle, serine at the C-terminus.

Energy Cost

Forming a peptide bond is thermodynamically unfavorable (positive ΔG) under cellular conditions. Your ribosome pays for it: each new amino acid costs 4 high-energy phosphate bonds (1 ATP to charge the tRNA with its amino acid, plus 2 GTP during elongation and 1 GTP during translocation). That is why protein synthesis is among the most energy-expensive processes in the cell.

Breaking Peptide Bonds: Hydrolysis

The reverse reaction breaks a peptide bond by adding water back. Your stomach acid and digestive enzymes (pepsin, trypsin, chymotrypsin) all catalyze peptide bond hydrolysis. Without an enzyme, the reaction is glacially slow - peptide bonds have half-lives of hundreds of years in neutral water. With the right enzyme, it happens in milliseconds.

Specificity: Why Sequencing Works

Not all proteases are created equal. Each one recognizes specific amino acid residues at the cleavage site, which is what makes peptide mapping and sequencing possible. If you already know the cutting rules, you can predict exactly which fragments a protease will generate from a given sequence.

Peptide Length Terminology

• Dipeptide: 2 amino acids, 1 peptide bond
• Tripeptide: 3 amino acids, 2 peptide bonds
• Oligopeptide: a few (usually <10-20) amino acids
• Polypeptide: longer chain, but not yet folded or functional
• Protein: one or more polypeptide chains folded into a functional 3D shape

Note that for n amino acids, there are always (n - 1) peptide bonds.

A peptide bond looks like a single C-N bond. It isn’t. Two electron resonance structures share the electrons between a C-N and a C=N form. The result is partial double bond character, which has three consequences you need to know cold:

1. The peptide bond is planar (all six atoms in the amide group lie in a flat plane).
2. It is rigid (no rotation around the peptide bond itself).
3. The substituents on either side adopt the trans configuration in almost every case.

Everything about how proteins fold starts from these three facts.

Why the Peptide Bond is Planar

The nitrogen in an amide has a lone pair. That lone pair delocalizes into the neighboring carbonyl C=O. The resonance structure puts a double bond between C and N and a negative charge on the oxygen. In the real molecule, this is an average: the C-N bond has about 40 percent double-bond character.

Double bonds cannot rotate. Therefore the peptide bond cannot rotate. All six atoms involved - the alpha carbon of residue i, the carbonyl C and O, the amide N and H, and the alpha carbon of residue i+1 - lie in a single plane.

Why Trans is Preferred

When two alpha carbons are bonded through a planar peptide bond, the R groups can either be on the same side of the plane (cis) or opposite sides (trans). Trans is strongly preferred because it keeps the bulky R groups away from each other. Cis peptide bonds cause steric clashes.

About 99.9 percent of peptide bonds in folded proteins are trans. The rare exception: proline. Because proline’s side chain loops back to the amide nitrogen, the cis and trans forms are closer in energy, and about 5 to 10 percent of X-Pro peptide bonds (peptide bond immediately before proline) are cis.

Where the Backbone Can Rotate: Phi and Psi

If the peptide bond itself is frozen, where does the flexibility come from? The two bonds on either side of each alpha carbon. These are called phi (Φ) and psi (Ψ):

• Phi (Φ): the rotation around the N-Cα bond.
• Psi (Ψ): the rotation around the Cα-C(=O) bond.

Every residue in a polypeptide has its own Φ and Ψ. Together, these two angles for every residue define the entire backbone geometry of the protein.

The Ramachandran Plot

Because of steric clashes between the R group and the backbone, not every combination of Φ and Ψ is physically possible. Plotting Φ vs. Ψ for all residues in known protein structures gives the Ramachandran plot. Only a few clusters of allowed angles appear:

• The alpha helix region (around Φ = -60°, Ψ = -45°)
• The beta sheet region (around Φ = -120°, Ψ = +120°)
• A small left-handed helix region (mostly occupied by glycine)

The primary structure is the simplest thing in biology and the most important. It is just the order of amino acids in the chain, written from N-terminus to C-terminus. Nothing more.

Yet that one-dimensional string of letters determines everything: how the protein folds, what it binds, how fast it works, how long it lasts. Change one letter and you can destroy the whole protein - or, occasionally, build a new one.

How the Primary Structure is Specified

The gene dictates the primary sequence through the genetic code. Each codon (3 bases of mRNA) corresponds to one amino acid. The ribosome reads codons left to right and adds amino acids to the growing chain N-terminus first. Once translation finishes, the entire primary structure is set - nothing can change it without breaking a peptide bond.

Why Primary Structure is Hierarchically the Most Important

All other levels of structure are consequences of primary structure. The sequence “causes” the folding. Change the sequence and you change (potentially) everything downstream.

This is why biochemists say “the primary structure determines the tertiary structure.” Christian Anfinsen’s famous 1961 experiment showed this directly: ribonuclease was denatured (unfolded), then allowed to refold on its own in water. It regained its full enzymatic activity without any cellular help. The sequence carried all the information needed to fold.

Single Amino Acid Substitutions: Sickle Cell

The beta chain of adult hemoglobin is 146 amino acids long. In sickle cell disease, position 6 is changed from glutamate (E, negatively charged, hydrophilic) to valine (V, nonpolar, hydrophobic). One letter in 146. The rest of the sequence is identical.

Consequences of that single swap:

• Valine on the surface creates a hydrophobic sticky patch.
• At low oxygen, these hydrophobic patches on neighboring hemoglobin molecules stick together.
• The hemoglobin polymerizes into long rigid fibers.
• The fibers warp red blood cells into the characteristic sickle shape.
• Sickled cells clog capillaries and cause ischemic pain, organ damage, and anemia.

Determining Primary Structure in the Lab

Historically proteins were sequenced by Edman degradation: the N-terminal residue is cleaved off one at a time, identified by chromatography, and the process repeats. This is slow.

Modern methods use mass spectrometry. A protease (like trypsin) cuts the protein at specific residues (trypsin cuts after K or R), the fragments are analyzed by mass spec, and the sequence is reconstructed. A whole proteome can be identified this way.

After translation, the polypeptide chain begins to fold as it leaves the ribosome. The first folds are local - not the whole protein collapsing, just small regions snapping into repeating patterns. These local patterns are secondary structure, and they come in two flavors: alpha helices and beta pleated sheets.

Both are held together by the same force: hydrogen bonds between backbone amide groups. Not R groups. The backbone N-H of one residue hydrogen bonds to the backbone C=O of another residue a few positions away. R groups do not participate - they stick out to the side.

The Alpha Helix

The alpha helix is a right-handed corkscrew. The backbone spirals around an imaginary central axis. Every residue’s carbonyl oxygen (C=O) hydrogen bonds to the amide N-H exactly four residues ahead in the chain. Side chains project outward, away from the helix core.

Key numbers to know:

• 3.6 residues per turn
• 5.4 Å (0.54 nm) per turn - so 1.5 Å rise per residue
• Hydrogen bond is from residue n to residue n+4
• Most alpha helices are right-handed

Helix Breakers

Two amino acids disrupt alpha helices:

• Proline has a ring that locks its Phi angle and removes the amide N-H needed for hydrogen bonding. Proline almost never appears inside a helix (only at the N-terminal “cap” position).
• Glycine is too flexible. Its side chain is just hydrogen, so it samples too many conformations. It prefers turns and loops over the regular helix geometry.

The Beta Pleated Sheet

Beta sheets form between beta strands - segments of backbone running in an extended zigzag. Two or more strands line up next to each other and form hydrogen bonds sideways between adjacent strands. The result is a pleated sheet that looks like an accordion.

Beta sheets come in two flavors:

• Parallel: both strands run in the same N-to-C direction. Hydrogen bonds are slightly skewed. Less stable than antiparallel.
• Antiparallel: strands run in opposite N-to-C directions. Hydrogen bonds are linear (straight across). More stable.

Beta strands are separated in sequence but close in space. Between two adjacent strands in a sheet, there must be a connecting loop or “beta turn.”

Loops and Turns

Not every residue participates in a helix or sheet. In between, the backbone makes turns (short reversals of direction, often 3-4 residues) and loops (longer irregular segments). These regions often sit on the protein surface and contain active sites, binding loops, or antibody recognition loops.

Glycine and proline are over-represented in loops and turns. Glycine provides flexibility; proline enforces a sharp kink.

Why Secondary Structure Exists

Hydrogen bonding the backbone amides is energetically cheap and there are a lot of them in a polypeptide. If the protein did NOT form secondary structure, those H-bond donors (N-H) and acceptors (C=O) would need to find other partners - usually water. Burying the backbone in the protein interior requires first satisfying all those H-bonds internally. That is exactly what alpha helices and beta sheets do.

Primary structure is a flat sequence. Secondary structure is local folding (helices and sheets). Tertiary structure is the whole polypeptide chain folded into its final 3D shape, with the helices and sheets packed against each other and a stable core in the middle.

Tertiary structure is where the protein becomes functional. An enzyme’s active site is a tertiary feature. A binding pocket on a hormone receptor is a tertiary feature. If tertiary structure is wrong, the protein does not work.

The Five Forces That Stabilize Tertiary Structure

Hydrophobic Interactions: the Main Engine

Nonpolar R groups cannot hydrogen bond with water. When a protein folds, exposed nonpolar residues disrupt the water’s hydrogen bond network, forcing water molecules to organize into “cages” around them (entropy penalty). Burying those nonpolar residues in a protein interior releases those ordered water molecules back into the bulk. That entropy gain is the driving force of folding.

Disulfide Bonds

Two cysteines can be oxidized to form a covalent -S-S- bridge. This is the strongest single bond stabilizing tertiary structure - a true covalent bond, not a weak interaction.

Disulfide bonds require an oxidizing environment. Inside the cytoplasm (reducing environment), they rarely form. In the endoplasmic reticulum and in extracellular space (oxidizing environments), they are common. That is why secreted proteins (insulin, antibodies, digestive enzymes) tend to be rich in disulfide bonds while cytosolic proteins are not.

Salt Bridges (Ionic Interactions)

Salt bridges form between a negatively charged side chain (Asp or Glu) and a positively charged one (Lys, Arg, His). They are weaker than covalent bonds but can have a significant effect on stability when buried inside the protein where no water can interfere.

Salt bridges are pH-sensitive. Lowering pH (protonating Asp/Glu) or raising pH (deprotonating Lys/Arg) removes the charge and breaks the bridge - one reason proteins denature at extreme pH.

Hydrogen Bonds and Van der Waals

Polar side chains (Ser, Thr, Tyr, Asn, Gln, His) can hydrogen bond with each other or with the backbone. Van der Waals forces (instantaneous dipoles) act between all atoms when they are close enough. Individually weak, but thousands of van der Waals contacts in a tightly packed protein interior add up.

Some proteins are lone wolves - one polypeptide chain folds, does its job, and that is the whole story (myoglobin, for example). Other proteins are teams - two or more polypeptide chains assemble into a functional complex. That team assembly is the quaternary structure.

Only proteins with more than one polypeptide chain have a quaternary structure. A single-chain protein has primary, secondary, and tertiary structure. Add a second chain and you have quaternary.

The Vocabulary

• Monomer: single polypeptide chain.
• Dimer: two subunits. A homodimer has two identical subunits; a heterodimer has two different ones.
• Trimer: three subunits (e.g., collagen is a triple helix).
• Tetramer: four subunits (e.g., hemoglobin).
• Oligomer: general term for any multisubunit complex.

Hemoglobin: the Classic Example

Adult hemoglobin is a heterotetramer of two alpha chains and two beta chains (α2β2). Each subunit has its own tertiary structure and binds one heme group. The four subunits fit together in a compact roughly tetrahedral arrangement.

Why Quaternary Structure Matters

Two big reasons quaternary structure shows up all over biology:

1. Cooperativity. When one subunit binds a ligand, it can change its shape and transmit that change to the others, making them more (or less) eager to bind. Hemoglobin does this with oxygen: binding O2 at one subunit pulls the other three into their high-affinity state. This is why the hemoglobin-oxygen curve is sigmoidal while single-subunit myoglobin’s curve is hyperbolic.

2. Allosteric regulation. A molecule can bind at one site and affect activity at a distant site on another subunit. This is how many enzymes are controlled. Multisubunit proteins have more surface area and more interfaces for regulatory molecules to latch onto.

Subunit Interfaces Use the Same Forces as Tertiary Structure

Quaternary assembly uses the same bond types that build tertiary structure: hydrophobic contacts at the subunit interface, ionic interactions, hydrogen bonds, and sometimes disulfide bonds that bridge two chains (like the inter-chain disulfides in insulin and immunoglobulins). There is no new chemistry at the quaternary level - just a bigger playing field.

A polypeptide emerges from the ribosome as a loose chain. Over seconds to minutes, it folds into a specific 3D shape called the native state. That folded structure is what does the job - whether the job is catalyzing a reaction, carrying oxygen, or supporting a cell.

Break the native shape and the protein stops working. Breaking the shape is called denaturation. Sometimes denatured proteins can refold. Often they cannot.

The Native State

The native state is the conformation where the protein has its lowest free energy under physiological conditions. Usually this corresponds to the functional fold. The native state is:

• Stable enough to survive normal cellular conditions
• Often flexible enough to allow catalysis or conformational changes
• Not always the most thermodynamically stable form possible - some proteins fold into metastable, kinetically trapped states, especially membrane proteins and amyloids

Folding happens in milliseconds to seconds for most proteins, which is remarkable given how many possible conformations a 150-residue chain could explore.

The Thermodynamics of Folding

Folding looks like order emerging from disorder, which seems to violate the second law of thermodynamics. It does not - you just have to count the water.

The governing equation is ΔG = ΔH - TΔS. For folding to be spontaneous, ΔG must be negative. Here is what each term actually contributes:

This is the hydrophobic effect, and it is the #1 driver of folding. It explains why:

• Hydrophobic residues end up buried in the protein core
• Polar and charged residues stay on the surface, where they contact water
• Heating can denature a protein (heat raises TΔS for the unfolded state even more, tipping the balance)

Chaperones

Inside cells, many proteins cannot fold on their own - the cytoplasm is too crowded and some intermediates have exposed hydrophobic patches that would stick to neighboring proteins and aggregate. Chaperone proteins bind to newly synthesized polypeptides and keep them isolated while they fold.

The main chaperone families (no need to memorize the exact names):

• Hsp70 family - binds nascent chains, prevents aggregation
• Hsp60 / GroEL-GroES family - provides an isolated barrel-shaped chamber where a single polypeptide can fold

What Denatures a Protein

Denaturation is the loss of the folded 3D structure. The primary sequence (covalent peptide bonds) is almost always preserved - only the weak forces that stabilize folding get disrupted.

| Agent | Mechanism |
|-------|-----------|
| Heat | Vibrations overcome hydrogen bonds and hydrophobic packing |
| Low pH (acid) | Protonates carboxyls, disrupts salt bridges, protonates histidines |
| High pH (base) | Deprotonates amines and tyrosines, disrupts salt bridges |
| Urea and guanidinium chloride | Chaotropes - compete for H-bonds with water, destabilizing the hydrophobic effect |
| Detergents (SDS) | Coat the protein surface, break hydrophobic contacts |
| Reducing agents (β-ME, DTT) | Break disulfide bonds |
| Heavy metals | Bind to cysteines, ionic groups, disrupt folding |
| Mechanical agitation | Exposes hydrophobic interior (whipping egg whites) |

Denaturation: Reversible or Irreversible?

Sometimes denatured proteins spontaneously refold when the denaturant is removed. Ribonuclease is the classic case - Anfinsen showed that ribonuclease denatured by urea and DTT refolded to full activity when the chemicals were washed out. The sequence held all the information.

More often, denaturation is effectively irreversible:

• Egg whites (fried or whipped) cannot be un-denatured
• Boiled milk cannot refold its proteins
• Most denatured proteins aggregate before they get a chance to refold, because exposed hydrophobic patches stick together

Many proteins cannot do their job with just 20 amino acids. They recruit a non-protein helper - a metal ion, a vitamin derivative, a sugar chain, a heme group. A protein that carries a non-protein component is called a conjugated protein.

The protein part is the apoprotein (without the helper). Together with the helper, it is the holoprotein (the complete, functional form). If the apoprotein cannot function alone, the helper is essential - and the helper itself gets a name depending on how tightly it binds.

Prosthetic Groups vs. Cofactors vs. Coenzymes

Three related terms that get confused:

• Cofactor: any non-protein helper. Includes both metal ions and organic molecules.
• Coenzyme: an organic cofactor. Most are vitamin-derived (NAD+, FAD, CoA, biotin).
• Prosthetic group: a cofactor that is permanently and tightly bound to the protein (often covalently). Heme is a prosthetic group.

The simple way to keep this straight: prosthetic group = glued on; cofactor = the general category; coenzyme = an organic cofactor that may bind loosely.

Major Types of Conjugated Proteins

Hemoproteins

Heme is an iron ion held inside a porphyrin ring. It is a prosthetic group in:

• Hemoglobin and myoglobin - reversibly bind O2
• Cytochromes - shuttle electrons in the electron transport chain
• Catalase - breaks down hydrogen peroxide

All heme proteins use the iron’s ability to switch between Fe2+ and Fe3+ (cytochromes) or to hold Fe2+ in a special “ready” state for O2 binding (hemoglobin/myoglobin).

Glycoproteins

A carbohydrate chain (often branched) is attached to a serine, threonine, or asparagine side chain. Glycoproteins are everywhere:

• Cell-surface receptors and adhesion molecules
• Antibodies (IgG is about 4 percent carbohydrate)
• Blood-type antigens (ABO antigens are glycolipid/glycoprotein differences)
• Most secreted proteins have sugar decorations

Lipoproteins

Proteins that wrap around a core of cholesterol and triglycerides to transport lipids through the aqueous blood. The major classes (HDL, LDL, IDL, VLDL, chylomicrons) differ in lipid-to-protein ratio, which affects their density. More lipid = less dense. You will see these in detail in the lipid chapter.

Metalloproteins

A metal ion is held in the active site by side chain ligands (usually histidine, cysteine, aspartate, or glutamate). Metal ions catalyze reactions that the 20 amino acids alone cannot - redox chemistry (Fe, Cu), Lewis-acid catalysis (Zn), and structural stabilization (Mg, Zn fingers in transcription factors).