Wyatt Technology has published an application note demonstrating the capability of its Dynapro dynamic light scattering (DLS) instrument.
The note details the instrument's capabilities in monitoring the dissociation of an oligomeric protein and unfolding of monomers simultaneously using chaotropic agents.
The analysis of proteins is important for the study of many diseases such as cancer, diabetes, cardiovascular disease and neurodegenerative diseases such as Alzheimer's.
The importance of protein folding and unfolding has long been recognised and is an active field of research.
The main rules of protein folding/unfolding have been established from in vitro experiments and the information obtained can be related to the function and behaviour of proteins in vivo.
Unfolding of proteins in vitro can be induced chemically using chaotropic agents like guanidine hydrochloride or urea.
Protein Y is a 16 kDa, almost fully helical human protein.
At pH 5.0 it forms oligomers of different sizes.
Studying the unfolding of protein Y by circular dichroism, the main transition in the secondary structure can be observed.
The aim of this specific study was to follow the unfolding process by another independent method.
For this application, far UV CD spectra were taken at 25C on an Aviv spectrophotometer using a 0.01cm cell.
Five spectra were recorded with a step size of 1.0nm, a bandwidth of 1.5nm and an averaging time of 5s.
Samples were prepared three hours before measurement to allow time for the unfolding.
The protein concentration was 2 mg/ml.
The inflexion point of the unfolding curve is at 2.6 M GnHCl by both methods.
DLS was performed with a Dynapro using a 12ul quartz cuvette.
Samples were filtered with a 0.01um Whatman Anodisc-13 filter.
Data were analysed with Wyatt Dynamics software and 25-30 acquisitions were averaged.
The main transition in secondary structure can be observed by estimating the Stokes radius of the protein under different concentrations of the chaotropic agent.
The mid-point of the transition agrees exceptionally well with the CD data (2.6M GnHCl).
However, by applying dynamic light scattering measurements it is possible to observe additional molecular events: the dissociation of oligomers in the 0-2 M GnHCl concentration range.
The results of the experiment demonstrate that dynamic light scattering measurements are capable of quickly and easily monitoring the dissociation of an oligomeric protein and unfolding of monomers simultaneously using chaotropic agents.
