Biology subjects

Kjaergaard, M.

Publications and source records attributed to Kjaergaard, M..

4 recordsLinked to original sources

bioRxiv2019

Helix formation during the coupled binding and folding of intrinsically disordered proteins monitored by synchrotron-radiation circular dichroism spectroscopy

Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signalling processes. These proteins often gain structure upon binding to their target proteins in multi-step reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain (NCBD) and the disordered interaction domain from activator of thyroid hormone and retinoid receptors (ACTR). We demonstrate that helix formation in a rapid binding reaction can be followed by stopped flow synchrotron-radiation circular dichroism spectroscopy, and describe the design of such a beamline. Fluorescence-monitored binding experiments of ACTR and NCBD display several kinetic phases including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time resolved circular dichroism experiments show that almost all helicity is formed upon initial association of the proteins, or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron radiation circular dichroism for mechanistic studies of protein-protein interactions.

biophysics↗

bioRxiv2019

Nanoscale spatial dependence of avidity in an IgG1 antibody

Antibodies are secreted proteins that are crucial to recognition of pathogens by the immune system and are also efficient pharmaceuticals. The affinity and specificity of target recognition can increase remarkably through avidity effects, when the antibody can bind a multivalent antigen through more than one epitope simultaneously. A key goal of antibody engineering is thus to optimize avidity, but little is known about the nanoscale spatial dependence of avidity in antibodies. Here, we develop a set of anti-parallel coiled-coils spanning from 8-21 nm and validate their structure using biophysical techniques. We use the coiled-coils to control the spacing between two epitopes, and measure how antigen spacing affects the stability of the bivalent antibody:antigen complex. We find a maximal avidity enhancement at a spacing of 14 nm, but only see a [~]2-fold variation of avidity in the range from 8-21 nm. In contrast to recent studies, we find the avidity to be relatively insensitive to epitope spacing near the avidity maximum as long as it is within the spatial tolerance of the antibody. The coiled-coil systems developed here may prove a useful protein nanocaliper for profiling the spatial tolerance and avidity profile of bispecific antibodies.

biophysics↗

bioRxiv2019

Linker dependence of avidity in multivalent interactions between disordered proteins

Multidomain proteins often interact through several independent binding sites connected by disordered linkers. The architecture of such linkers affect avidity by modulating the effective concentration of intra-molecular binding. The linker dependence of avidity has been estimated theoretically using simple physical models, but such models have not been tested experimentally since the effective concentrations could not be measured directly. We have developed a model system for bivalent protein interactions connected by disordered linkers, where the effective concentration can be measured using a competition experiment. We characterized the bivalent protein interactions kinetically and thermodynamically for a variety of linker lengths and interaction strengths. In total, this allowed us to critically assess the existing theoretical models of avidity in disordered, multivalent interactions. As expected, the onset of avidity occurs when the effective concentration reached the dissociation constant of the weakest interaction. Avidity decreased monotonously with linker length, but only by a third of what is predicted by theoretical models. We suggest that the length dependence of avidity is attenuated by compensating mechanisms such as linker interactions or entanglement. The direct role of linkers in avidity suggest they provide a generic mechanism for allosteric regulation of disordered, multivalent proteins.

biophysics↗

bioRxiv2019

Effective concentrations enforced by intrinsically disordered linkers are governed by polymer physics

Many multidomain proteins contain disordered linkers that regulate inter-domain contacts, and thus the effective concentrations that govern intra-molecular reactions. Effective concentrations are rarely measured experimentally and therefore little is known about how they relate to linker architecture. We have directly measured the effective concentrations enforced by disordered protein linkers using a new fluorescent biosensor. We show that effective concentrations follow simple geometric models based on polymer physics, offering an indirect method to probe the structural properties of the linker. The compaction of the disordered linker depends not only on net charge, but also on the type of charged residues. In contrast to theoretical predictions, we found that polyampholyte linkers can contract to similar dimensions as globular proteins. Hydrophobicity has little effect in itself, but aromatic residues lead to strong compaction likely through {pi}-interactions. Finally, we find that the individual contributors to chain compaction are not additive. This work represents perhaps the most systematic study of the relationship between sequence and structure of intrinsically disordered proteins so far. A quantitative understanding of the relationship between effective concentration and linker sequence will be crucial for understanding disorder-based allosteric regulation in multidomain proteins.

biophysics↗