Studying intermolecular protein associations with FIDA: a conversation with Prof. Daniel Otzen

Prof. Daniel Otzen works at iNANO, Aarhus University, across two sides of protein science: the harmful protein aggregation behind diseases such as Parkinson's, and enzymes put to useful work, from operating at low temperatures to breaking down plastic. Different as they are, one instrument has become central to both. In a conversation with Fidabio, Prof. Otzen explained how Flow Induced Dispersion Analysis (FIDA) serves that range of work. This is a summary of what he said.

Why Prof. Otzen relies on FIDA for oligomers

A central question in the Otzen lab's Parkinson's work is what makes some protein aggregates harmful. Large insoluble aggregates may simply sit in the cell, but small soluble complexes, oligomers, move quickly and interact with many things, and are potentially the more dangerous species.

"Aggregates can be insoluble when they accumulate in the cell, and then they're just large and lie around and don't do much. But they can also be small soluble complexes that we call oligomers. They can move around quite quickly and interact with a lot of things," Prof. Otzen said.

Those oligomers are hard to study: only small amounts can be made, they must not be perturbed by binding to a surface, and true equilibrium conditions are needed. FIDA meets all three at once, which is why it became central here.

"The oligomers can be made in the lab, but you can't make a lot of them. So you need to have a technique that can work with small concentrations and small volumes, and the FIDA is second to none there," Prof. Otzen said.

"You also want to work with it under conditions where you don't manipulate these oligomers, you don't bind them to a surface or immobilize them. FIDA is really great here because it allows you to have it in solution and then expose it to other components. It has to be under truly equilibrium conditions, and all this is something that FIDA provides," Prof. Otzen said.

Telling binding apart from a functional effect

Prof. Otzen calls FIDA a game changer for understanding how oligomers engage membranes and other partners, because it measures robust equilibrium affinity constants for the binding itself, separate from what the binding then does.

"It allows you to measure, correct or robust equilibrium affinity constants for how oligomers can bind to whatever you expose it to," Prof. Otzen said.

"We know that they can bind to membranes and lead to disruption of them, so that things can leak out. But there is a difference between binding and leakage. And by having the FIDA technique, you can actually directly measure if the oligomer is binding to the membrane or not. That is really useful," Prof. Otzen said.

Enzyme assembly and activity from size

The same size readout serves the lab's enzyme work, where a protein may exist as a monomer, dimer or tetramer, each with potentially different activity. Because FIDA reports size down to very low concentrations, the group can follow how concentration shifts that assembly state and, in turn, catalytic behaviour.

"FIDA is really great because, of course, it provides information about the size, and you can really go down in extremely low concentrations to find out how their concentration affects the association of the protein," Prof. Otzen said.

Specificity in complex mixtures

A further strength is following an interaction directly inside a complex biological sample. With only the protein of interest fluorescently labelled, everything else stays invisible to the detector, so the measurement reports on that protein alone even in a crowded background.

"You just need to have a fluorescently labeled protein of interest, and then you can expose it to a very complex mixture and see whether it binds to something in that mixture. That mixture will be invisible in FIDA because the components are not fluorescently labeled. We've actually done that to show, for example, how the oligomer can bind to different components in a cell lysate, where there can be thousands of different components," Prof. Otzen said.

FIDA does not name the binding partner, but, as Prof. Otzen put it, "it simply provides readouts under realistic physiological conditions," which then guide the next experiments.

Screening biologics at scale

That economy pays off in larger studies. In recent work the lab tested how more than 30 monoclonal antibodies interact with a protein oligomer, ranking them rigorously under identical conditions.

"We looked at more than 30 different monoclonal antibodies and how they interact with the oligomer. And we could measure all of their interactions at no particular cost of either antibodies or oligomer, simply because FIDA is such an economical technique. We can also compare them rigorously with each other under exactly the same conditions. That has also really made a difference for us in our understanding and our ranking of these different antibodies," Prof. Otzen said.

"I believe that FIDA could really become a basically standard technique within the pharmaceutical industry to assay interactions between biologics and their targets," Prof. Otzen said.

What Prof. Otzen values most

Asked what he would miss most if FIDA disappeared, Prof. Otzen pointed to the clarity of the readout.

"What I really like about FIDA is that you get a very understandable readout. You get the hydrodynamic radius. Many techniques only provide an indirect readout, so FIDA's very straightforward and I think understandable and also testable outcome," Prof. Otzen said.

"The fact that it's under equilibrium conditions at little cost, and you've got high throughput, or at least medium throughput options, they all help out. The fact that you get a truly molecular picture of what's going on is unique for FIDA," Prof. Otzen said.

Where the work is heading

Looking ahead, Prof. Otzen described wanting to turn FIDA on enzymes that break down plastic. After reducing plastic to small, suspendable particles, the aim is to follow their depolymerisation, the size reduction of the pieces, directly on FIDA. He also flagged interest in the kinetics package, since the work so far has been at equilibrium and following slow association and dissociation steps is a natural next step.

"I'm really keen to see how we can monitor the depolymerization or the sort of increased size reduction of these small plastic pieces by FIDA in a very direct fashion," Prof. Otzen said.

What we learned from this conversation

Across the Otzen lab's two worlds, disease-linked protein aggregation and useful enzymes, the same qualities keep FIDA central: it works with the tiny amounts of fragile oligomers the group can make, in solution and at equilibrium so nothing is immobilised, and it returns a direct, understandable hydrodynamic-radius readout rather than an indirect signal. That let Prof. Otzen separate binding from membrane leakage, read enzyme assembly state from size, follow an oligomer's interactions inside raw cell lysate, and rank more than 30 antibodies under identical conditions at little cost, and it is now heading toward plastic-degrading enzymes and kinetics. It is, in his view, a uniquely molecular picture of what is going on.

You can find publications from Prof. Otzen's laboratory, and from other FIDA users, in the peer-reviewed literature.

Curious how FIDA measures binding, size and aggregation in solution? Explore the FIDA technology and the Fida instrument, or browse the peer-reviewed literature. Read more scientists’ experiences with FIDA.