Protein structure and purification, exploring PDB structures, enzyme kinetics and inhibition, binding and thermodynamics, spectroscopy, structural biology, molecular simulation and research career routes.

Biochemistry & Biophysics

## Why it matters

Proteins do most of the work in a cell: enzymes catalyse reactions, antibodies recognise invaders, transporters move ions, and structural proteins hold tissues together. What a protein does depends on its three-dimensional shape, and that shape is encoded in its amino acid sequence. Biochemists and biophysicists spend careers measuring, predicting and changing protein structure, so the four levels of structure are the vocabulary for everything else in this course.

## Building blocks: amino acids and the peptide bond

Proteins are chains of the 20 standard **amino acids**. Each has the same backbone (an amino group, a central alpha carbon and a carboxyl group) and a different **side chain** (R group). Side chains are often grouped by chemistry:

- **Nonpolar / hydrophobic:** Ala, Val, Leu, Ile, Met, Phe, Trp, Pro. They tend to be buried inside the folded protein.
- **Polar, uncharged:** Ser, Thr, Asn, Gln, Tyr, Cys. They can form hydrogen bonds.
- **Charged at pH 7:** Asp and Glu (negative); Lys and Arg (positive); His is partly positive near neutral pH.
- **Special cases:** Gly is tiny and flexible; Pro locks the backbone and breaks helices; Cys can form **disulfide bonds**.

Amino acids join by **peptide bonds**, formed between the carboxyl of one and the amino group of the next. The peptide bond has partial double-bond character, so it is flat and rigid. The backbone can rotate only around two bonds per residue, described by the **phi (φ) and psi (ψ) angles**. A **Ramachandran plot** of φ against ψ shows which combinations are allowed without atoms clashing.

## The four levels of structure

**1. Primary structure** is the amino acid sequence, written from the N-terminus to the C-terminus, such as `MKTAYIAKQR...`. It is held together by covalent peptide bonds and is read directly from the gene (or measured by mass spectrometry).

**2. Secondary structure** is the local folding of the backbone, stabilised by **hydrogen bonds between backbone C=O and N–H groups**:

- **Alpha helix:** a right-handed coil with 3.6 residues per turn; each C=O bonds to the N–H four residues along.
- **Beta sheet:** extended strands lying side by side, hydrogen bonded between strands, either parallel or antiparallel.
- **Turns and loops** connect them and are often where function happens.

**3. Tertiary structure** is the overall 3D fold of one polypeptide chain. It is driven mainly by the **hydrophobic effect** (nonpolar side chains cluster away from water) and stabilised by hydrogen bonds, ionic interactions (salt bridges), van der Waals contacts and, in many secreted proteins, disulfide bonds. Recurring folded units are called **domains**.

**4. Quaternary structure** is the arrangement of several chains (subunits) into one complex. Hemoglobin, for example, has four subunits (two alpha, two beta), and its subunits shift relative to each other when oxygen binds, which is the basis of cooperative binding.

Not every protein has quaternary structure, and some proteins or regions have no stable fold at all: **intrinsically disordered** regions are common and functionally important.

## Worked example: reading a structure description

A database entry says: "Homodimer; each chain has an N-terminal Rossmann-fold domain (alternating alpha helices and parallel beta strands) and a C-terminal catalytic domain; one disulfide per chain."

- Quaternary: two identical chains (homodimer).
- Tertiary: two domains per chain.
- Secondary: helices and parallel beta strands in the first domain.
- Covalent extras: a disulfide bond within each chain.

## Folding and stability

Anfinsen's experiments on ribonuclease showed that a denatured protein can refold on its own, so the sequence contains the information for the fold. In the cell, **chaperones** help many proteins fold and prevent aggregation. A folded protein is only marginally stable: the free energy difference between folded and unfolded states is often just 20–60 kJ/mol, the size of a few hydrogen bonds. Heat, extreme pH, detergents and chemical denaturants (urea, guanidinium) can unfold it. **Misfolding and aggregation** are linked to diseases such as Alzheimer's and Parkinson's.

## Common mistakes

- **Saying secondary structure comes from side-chain interactions.** It comes from backbone hydrogen bonds; side chains mainly drive tertiary structure.
- **Thinking disulfides hold most proteins together.** Many intracellular proteins have none; the hydrophobic effect is the main driver of folding.
- **Assuming one sequence means one rigid shape.** Proteins breathe, flex and often change conformation when they bind something.

## Recap

- Primary: sequence. Secondary: backbone hydrogen-bonded helices and sheets. Tertiary: whole-chain fold. Quaternary: multiple chains.
- The hydrophobic effect drives folding; many weak interactions stabilise it.
- Structure determines function, and small stability margins make proteins sensitive to their conditions.

## Try it

This lesson has no activity. Check your understanding with the quiz on the Do tab.


Explain the four levels of protein structure, what drives folding and why folded proteins are only marginally stable.

Protein structure

Protein Structure and Folding

Protein Structure - Primary, Secondary, Tertiary, & Quarternary - Biology

Choose a well-known enzyme (for example, T4 lysozyme or a bacterial beta-lactamase) and look up its molecular weight and isoelectric point in its UniProt entry (https://www.uniprot.org/).

Assume it is expressed in E. coli with an N-terminal His-tag. Write the steps from harvested cells to clarified lysate, and note what each step removes.

Pick a capture, an intermediate and a polishing step. For each, write the property it separates by, the binding and elution conditions, and why it follows the previous step.

Mark every point where you need a buffer exchange, and say why (for example, imidazole or salt that would stop binding in the next column).

Fill in a purification table for this data: lysate 1,500 mg and 6,000 U; IMAC 30 mg and 4,500 U; ion exchange 9 mg and 3,600 U; SEC 6 mg and 3,000 U. Compute specific activity, yield and fold purification for each step.

Decide which step adds the least purification for the yield it costs, and write one sentence on whether you would keep it.

List the quality checks you would run on the final sample (for example SDS-PAGE, A280 concentration, activity, SEC profile for aggregates).

If your teaching lab runs a purification practical, compare your plan with the lab protocol and ask your instructor about any differences.

## Why it matters

To study a protein's structure, kinetics or binding, you first need it pure: a sample where your protein is the main component and contaminants will not confuse the measurement. Purified proteins are also products, from research reagents to insulin and monoclonal antibodies. Planning a purification scheme, and judging it with a **purification table**, is a core biochemistry skill and a frequent interview question for research associate roles.

## Where the protein comes from

Most research proteins are **recombinant**: the gene is cloned into an expression host (often *E. coli*, yeast, insect or mammalian cells), usually with an added **affinity tag** to make purification easier. The cells are harvested and broken open (**lysis**) by sonication, high-pressure homogenisation or detergents, and the debris is removed by centrifugation to give a **clarified lysate**. All of this is done in a lab under supervision, following institutional biosafety rules for the organisms involved.

## Separating by properties

Each chromatography method separates proteins by a different property, so a good scheme combines methods that work on **different** properties:

- **Affinity chromatography** uses a specific binding partner. A **His-tag** (six or more histidines) binds nickel or cobalt ions on the resin (**IMAC**, immobilised metal affinity chromatography); you wash away other proteins and elute with imidazole. GST tags bind glutathione; antibodies bind Protein A. Affinity steps can give large purification in one step.
- **Ion exchange (IEX)** separates by **charge**. Below its isoelectric point (pI) a protein is positively charged and binds a cation exchanger; above it, it binds an anion exchanger. Elution uses a salt gradient.
- **Size exclusion chromatography (SEC, gel filtration)** separates by **size and shape**. Large molecules come out first. It is usually the last "polishing" step, because it also removes aggregates and swaps the protein into its final buffer.
- **Hydrophobic interaction chromatography (HIC)** separates by surface hydrophobicity, binding at high salt.

A classic order is **capture → intermediate → polish**: for example IMAC, then ion exchange, then SEC. Between steps you may need to change buffer (dialysis or desalting) because, for instance, the high salt or imidazole from one step will stop binding in the next.

## Checking each step

- **SDS-PAGE** shows which fractions contain a band at the expected molecular weight and how many contaminant bands remain.
- **Protein concentration** by A280 (using the protein's extinction coefficient) or a dye assay such as Bradford or BCA.
- **Activity assay** (for an enzyme) measures how much functional protein you kept.

## The purification table

The table tracks every step. For an enzyme:

- **Total protein** (mg).
- **Total activity** (units, U; for example µmol of product per minute).
- **Specific activity** = total activity / total protein (U/mg). It rises as the sample gets purer.
- **Yield** = total activity at this step / total activity in the lysate × 100 %.
- **Fold purification** = specific activity at this step / specific activity of the lysate.

**Worked example:**

```text
step            total protein (mg)   total activity (U)   specific (U/mg)   yield   fold
lysate          2000                 10000                5                 100 %   1
IMAC            40                   7000                 175               70 %    35
ion exchange    12                   5600                 467               56 %    93
SEC             8                    4800                 600               48 %    120
```

Each step loses some protein, so yield falls while purity rises. A step that raises fold purification very little but costs a lot of yield is a candidate for removal.

## Planning trade-offs

- **Stability:** keep the protein cold, at a pH and salt it tolerates, and add protease inhibitors to the lysis buffer. Some proteins need reducing agents or cofactors.
- **Tag removal:** a protease site between tag and protein lets you cut the tag off, then a second IMAC pass ("reverse IMAC") collects the tag and uncut protein, while your untagged protein flows through.
- **Scale and purpose:** a crystallography sample needs high purity and homogeneity; an antibody for a quick Western blot may need only one step. Therapeutic proteins are purified under GMP with validated processes.

## Common mistakes

- **Using two steps that separate by the same property** (such as two anion-exchange columns at the same pH).
- **Forgetting buffer compatibility** between steps.
- **Reporting purity without activity.** A clean band on a gel can still be inactive or aggregated.
- **Calculating yield from protein instead of activity.** Yield is about the target, so track the target's activity (or a specific measure such as a Western blot).

## Safety and real-world practice

This lesson is awareness level. Real purification uses biological materials, solvents and equipment such as centrifuges and high-pressure systems, and requires training and supervision in your institution's lab. The activity below is a paper exercise only.

## Recap

- Combine methods that separate by different properties: affinity, charge, size, hydrophobicity.
- Capture, intermediate, polish; check each step by SDS-PAGE and activity.
- Specific activity, yield and fold purification tell you whether a scheme works.

## Try it

On the Do tab, plan a purification scheme on paper and fill in a purification table.


Plan a capture, intermediate and polishing purification scheme and judge it with specific activity, yield and fold purification.

Protein purification

His-Tag Protein Purification theory & practice - Immobilized Metal Affinity Chromatography (IMAC)

Protein Purification

Open https://www.rcsb.org/structure/4HHB and write down the experimental method, the resolution, the number of chains and the ligands listed.

Open the validation report summary on the same page and note one quality indicator (for example clashscore or Ramachandran outliers) and how it compares with other entries.

Open the 3D view (Mol*). Colour the structure by chain and identify the two alpha and two beta subunits.

Switch to a cartoon representation and find an alpha helix and a loop. Note whether hemoglobin is mostly helix or mostly sheet.

Select one heme (HEM) ligand and show the residues around it. Find the histidine that coordinates the iron and write down its residue number and chain.

Use the measurement tool to measure the distance from the iron to the nearest nitrogen of that histidine, and record it in ångströms.

Look up human hemoglobin alpha (UniProt P69905) in the AlphaFold database (https://alphafold.ebi.ac.uk/) and note which regions have the highest and lowest pLDDT.

Write three sentences linking what you saw to function: where oxygen binds, why the protein has four subunits, and one thing the structure cannot tell you.

## Why it matters

More than 200,000 experimentally determined structures of proteins, nucleic acids and their complexes are freely available in the **Protein Data Bank (PDB)**, plus over 200 million predicted structures in the AlphaFold database. Before starting an experiment, a biochemist checks whether a structure of the protein (or a relative) exists, where the active site is, which residues contact a ligand, and how reliable the model is. Reading a structure entry and moving around it in a viewer is an everyday research skill.

## The PDB and its entries

The PDB is run by the worldwide PDB (wwPDB) partnership; the US portal is **RCSB PDB** at https://www.rcsb.org/ (Checked on 2026-10-23.). Each entry has a four-character **PDB ID**, such as `4HHB` (human deoxyhemoglobin), and a summary page listing:

- **Macromolecules:** each chain (A, B, C...) and what it is.
- **Ligands:** small molecules, ions and cofactors (heme, ATP, metal ions), each with a three-letter code.
- **Experimental method:** X-ray crystallography, cryo-EM or NMR.
- **Resolution** (for X-ray and cryo-EM) in ångströms (Å). Smaller is better: around 1.5 Å shows individual atoms clearly; around 3.5 Å shows the chain path and large side chains, with less certainty.
- **Validation report:** global quality indicators (for X-ray, R-work and R-free; Ramachandran outliers; clashscore) compared with other entries.
- The primary **citation** for the paper that reported it.

Coordinates download as mmCIF (the current standard) or the legacy PDB format: plain-text files listing each atom's position, occupancy and **B-factor** (how much the atom's position is smeared, often by motion).

## Experimental versus predicted

**AlphaFold DB** (https://alphafold.ebi.ac.uk/, Checked on 2026-10-23.) provides predicted models. Each residue has a **pLDDT** confidence score from 0 to 100: above 90 is very high confidence, below 50 often means a disordered region. Predicted models do not include ligands or show alternative conformations, and they are hypotheses to test, not measurements.

## Viewers

- **Mol\*** (Mol-star) runs in the browser on RCSB and PDBe pages. No install.
- **PyMOL** and **ChimeraX** are desktop programs widely used for figures and analysis. ChimeraX is free for non-commercial use; PyMOL has an open-source version and a licensed version.

Common viewer actions:

- Change **representation**: cartoon (shows secondary structure), sticks or ball-and-stick (atoms), surface (shape and pockets).
- **Colour** by chain, secondary structure, element, hydrophobicity or B-factor.
- **Select** residues or ligands and show what lies within a few ångströms.
- **Measure** distances and angles between atoms.

## Worked example: hemoglobin, 4HHB

On the RCSB page for 4HHB you will find:

- Two kinds of chain: alpha (chains A and C) and beta (chains B and D), so the quaternary structure is a heterotetramer.
- Ligand **HEM** (heme), one per chain, each with an iron atom.
- Method: X-ray diffraction, at a resolution of about 1.7 Å.

In the 3D viewer, colour by chain to see the tetramer. Focus on a heme: the iron sits in the flat porphyrin ring, and one histidine (the **proximal histidine**) coordinates the iron from one side. Because 4HHB is the deoxy form, no oxygen is bound on the other side. Measuring the iron to histidine nitrogen distance gives roughly 2 Å, a typical coordination bond length. You have just related a structure to its function: this is the site where oxygen binds.

## Reading quality critically

- Check resolution and the validation sliders before trusting fine details.
- Missing residues (gaps in the chain) usually mean flexible, disordered regions.
- High B-factors flag mobile or uncertain parts.
- A ligand's position in a low-resolution map can be ambiguous; look at the density fit if the viewer offers it.
- A crystal structure is one snapshot, possibly influenced by crystal packing or by the construct used (truncations, mutations, tags).

## Common mistakes

- **Treating a predicted model as experimental.** Check the source and pLDDT.
- **Ignoring the biological assembly.** The file may hold one copy (asymmetric unit) or several; RCSB shows the biologically relevant assembly separately.
- **Over-reading low resolution** side-chain positions or water molecules.

## Recap

- Use RCSB PDB entries for method, resolution, chains, ligands and validation.
- AlphaFold models come with per-residue confidence (pLDDT).
- In a viewer: change representation, colour, select and measure, and always check quality before drawing conclusions.

## Try it

On the Do tab, explore 4HHB in the free Mol* viewer in your web browser.


Read a Protein Data Bank entry and inspect a structure in a browser viewer, checking method, resolution and confidence.

Exploring structures

Exploring PDB Structures in 3D with MolStar (Mol*): Introductory Guide

The Protein Data Bank (PDB) - a user's guide

How proteins are built and folded, how a protein is purified and judged with a purification table, and how to explore an experimental structure in a viewer.