Observing Picomolar Protein Unfolding Using Resonance Light Scattering

TL;DR

This paper introduces a new method to detect protein unfolding at very low concentrations without using dyes, which is useful for studying protein stability in pharmaceuticals.

Contribution

The novel method enables label-free detection of protein melting transitions at picomolar concentrations.

Findings

The method successfully modeled BSA melting with a sharp Gaussian at 1 pM.

A time constant of 67 s was determined for the transient state during BSA melting.

The method detected BoNT-A melting in the presence of much more concentrated HSA.

Abstract

We here present a novel and sensitive methodology for determining the melting point (MP) of Bovine Serum Albumin (BSA) from micromolar to picomolar concentration levels under label-free conditions. At 1 pM we could model the melting with a sharp Gaussian. However, from the transient state observed during the melting process by using a simple exponential decay model, we determined a time constant of 67 s. We applied this methodology by studying a 3.3 pM sample of a botulinum toxin A (BoNT-A) (stabilized with 2.8 nanomolar denatured Human Serum Albumin (HSA)). We were able to determine the Tm of BoNT-A in the presence of approximately 1000-fold more concentrated HSA. This method enables the detection of protein melting transitions at picomolar concentrations without the use of a fluorescence dye. Its sensitivity and simplicity make it a valuable analytical tool for studying protein stability in diluted pharmaceutical formulations. This method is useful for correlating thermal conformational changes with catalytic function.

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