Mark_FisherProf Mark Fisher, Professor of Biochemistry and Molecular Biology at the University of Kansas Medical Center, recently spoke with Cambridge Healthcare Institute about the difficulty of understanding the causes of aggregation formation and the challenges in kinetic stability, and his strategy to assess ligand or solution-based stability of these aggregation-prone proteins. Dr. Fisher will be presenting this approach at the upcoming Protein Aggregates & Particles conference from 16-17 November, 2017, as part of the 9th Annual PEGS Europe Summit taking place in Lisbon, Portugal.

Dr. Mark Fisher received his BS degree in Chemistry from Purdue in 1982 and a Ph.D. in Biochemistry from the University of Illinois in 1987 under Dr. Stephen Sligar where he examined how cytochrome P450s biophysically controlled drug clearance rates by modulating electron transfer rates. He subsequently became a Postdoctoral fellow at NIH under Dr. Earl Stadtman until 1992 where he was among the pioneers in research to understand how molecular chaperones control protein folding and protein homeostasis in the cell. He is currently a full Professor in the Biochemistry and Molecular Biology Department at the University of Kansas Medical Center. His current research is focused on capturing or reversing protein misfolding/unfolding intermediates that can detected using various platforms of a chaperonin detection based biolayer interferometry. Applications include accelerating drug discovery pipelines that combines in silico drug design with validation of protein stability. In addition, Dr. Fisher has demonstrated that one can recapitulate endosomal environments to visualize real time pH-induced kinetics of toxin transitions using BLI technologies. In all cases, the overreaching goal of the laboratory is to identify methods to prevent; 1) protein unfolding events prior to aggregation, 2) protein misfolding that result in protein folding diseases and 3) protein toxin unfolding transitions that result in cell entry.


How has the industry changed or matured in terms of understanding the causes of aggregation formation and mechanisms of action?

The largest gray area in dealing with protein aggregation is the identification of regions that are susceptible toward transient unfolding and aggregation. Much progress has been made using newer faster isolation of protein fragments that pinpoint potential regions using hydrogen deuterium exchange measurements. This knowledge can lead toward specific engineering efforts to stabilize such regions and enhance protein stability for drug efficacy. Our implementation of nature’s guardians against protein aggregation to develop specific chaperonin biosensors has allowed us to enter the realm of detecting these transient, rapidly fluctuating regions even before large scale aggregation reactions are visible using more conventional particle dependent detection systems.

What are the challenges of kinetic stability of aggregation-prone proteins?

As stated above, the major challenge with protein therapeutics is developing stable proteins that can still function within biological systems. Proteins must remain flexible to a degree because protein dynamics and movement defines life. In more simple terms, increased protein stability can sometimes lead to a loss in biological activity. If stabilization can occur in regions that are not biologically relevant, than product stability can be quite helpful.

Can you describe your strategy to assess ligand or solution-based stability of aggregation-prone proteins using an automated chaperonin biolayer interferometry platform?

Our strategy is based on the observation that ligand based binding events translate into changes in protein dynamics. In this realm, one can observe instances where ligand binding will decrease flexibility and increase protein kinetic stability. Our chaperoin biolayer interferometry approach can also readily identify instances where molecular events will decrease protein kinetic stability. Our primary measurement depends on the amplification of the presence of hydrophobic aggregation prone patches on the protein surface that can be recognized by a very large promiscuous chaperonin. The automated approach depends on timed denaturant pulses where proteins that are specifically orientated on biosensor surfaces are momentarily exposed to denaturing conditions that are removed in a very precisely timed solution changes. The extent of denaturation directly correlates with the amplified signal from the chaperonin binding. If a stabilizer is present during the denaturation phase and delays the kinetic denaturation, the signal from the chaperonin binding is simply diminished. The resulting kinetically controlled denaturation isotherms are highly reproducible and overwhelming statistically significant. The beauty of this approach is that it works with any aggregation-prone protein that folds into a three dimensional structure.

To learn more about Dr. Fisher’s presentation and the PEGS Europe Summit, visit