A week in the life of a structural biologist

In structural biology, a cleanly purified protein is only one part of the puzzle. Ensuring optimum protein stability requires a combination of analytical techniques to fully understand the quality of your sample.

This case study follows one of our structural biologist customers navigating a project where the protein he had purified turned out to be significantly unstable, and illustrates how a multi-technique biophysical approach, combining ITC, DLS, DSF, and DSC, can uncover instability issues that conventional methods miss.

Day 1

Dave is a structural biologist characterizing a new protein target. He began his project by setting up a purification process, and after several months, had produced a usable amount of the protein. Size exclusion chromatography (SEC) showed a large monomeric peak, SDS-PAGE displayed a clear band at the correct mass, and MALDI-ToF analysis confirmed the mass.

Before performing X-ray crystallography, Dave set up an enzyme-based assay to ensure the protein was active – only to find that the results were inconsistent and highly variable.

In order to figure out whether the problem lay in the protein itself, the inhibitor, the substrate, or another component of the assay, Dave decides to use isothermal titration calorimetry (ITC) to probe the issue further upon the advice of a colleague. He prepares his sample for overnight dialysis and returns the following morning.

Watch this short video to find out more about the principles of isothermal titration calorimetry.

Day 2

The next day, Dave runs an ITC titration on the Microcal PEAQ-ITC, and the results do not resemble a textbook example (Figure 1, left). The data show weak signals, large errors in the binding parameters, and a stoichiometry value far below 1 (Figure 1, right). This is a clear indicator that a significant fraction of the protein is not competent for ligand binding, and that the sample quality, not the assay design, is the source of the variability.

Day 3

To understand the physical properties of his protein sample, Dave then uses dynamic light scattering (DLS) on the Zetasizer Advance. This confirms the presence of aggregated protein material (Figure 2). It appears the protein is sensitive to the conditions of the concentration step applied during purification, resulting in aggregation that was not detectable by SEC alone.

Day 4

With aggregation identified as the key issue, Dave aims to optimize his protein’s stability by screening a range of buffers using a thermal shift assay, to help identify conditions that improve protein stability.

Dave begins with a differential scanning fluorimetry (DSF) screen to assess protein thermal stability across multiple buffer conditions. However, his current instrument is unfit for this workflow because the unit relies strictly on extrinsic fluorescent dyes, the dye binds to the protein’s hydrophobic pockets—actively altering its stability and introducing a high risk of false positives or quenched signals.

While the screen flags Buffer #3 as a potential candidate, Dave cannot trust the data. Instead of delivering a rapid, walk-away answer, this clumsy extrinsic method forces him to hunt for an orthogonal technique just to verify his results, defeating the purpose of a high-throughput screen. To confirm the DSF results with a dye-free, label-free technique, Dave runs a targeted differential scanning calorimetry (DSC) experiment on the MicroCal PEAQ-DSC.

Unlike fluorescence-based methods, DSC directly measures changes in the heat capacity of the protein during thermal unfolding, providing a calorimetric signal. It also requires no labels or dyes, removing the risk of measurement artefacts introduced in the DSF step.

The PEAQ-DSC data corroborate the DSF result: Buffer #3 shows a large positive shift in apparent Tm, with the onset of unfolding occurring 22°C higher (Figure 3, green curve). Dave now has independent, quantitative confirmation that Buffer #3 stabilized his protein.

Returning to the PEAQ-ITC with his reformulated protein, Dave observes a markedly improved result with a stoichiometry value of N=1, confirming the buffer optimization (Figure 4). The protein is now fully competent for ligand binding.

With a stabilized and validated protein sample in hand, Dave re-runs the enzyme activity assay. The results are consistent and reproducible.

From one ITC experiment, Dave has derived the protein’s binding stoichiometry, binding affinity, and enthalpy, and knows that protein-ligand pairs that successfully pass ITC have higher chances for success in X-ray crystallography. Binding energetics obtained using ITC can be correlated with structural information.

Dave proceeds to crystallization, and subsequently determines the structure of both the free protein and its complex with the target ligand by X-ray crystallography.

With his project complete, Dave reviews the steps that led to a successful outcome, noting that a combination of analytical techniques allowed him to move from an unexplained assay failure to a structurally and biochemically characterized protein.

As he worked through the consumable inventory from the week, Dave considers how each technique required a different sample format, a different preparation protocol, and a different set of consumables. The scientific value of the multi-method approach was clear, but the operational overhead of using a different instrument for each analysis was considerable.

From his learnings this week, Dave realizes that an orthogonal and complementary biophysical toolkit is necessary to generate confident, reliable results. He realizes that each technique delivers one piece of the puzzle, and together, they form the full picture. If he were to improve his workflow for the next project, he would focus on higher throughput, lower consumable costs, and higher-quality data: the combination of which enables the right decisions to be made quickly and with confidence.

Related content:

• White paper: Understanding the colloidal stability of protein therapeutics using dynamic light scattering
• White paper: Developing a bioformulation stability profile
• Application note: Enhanced aggregate detection: monitoring protein stability using dual angle light scattering

Learn more about how ITC is used in drug discovery and development in our poster.