Jens Danielsson

Mutations in the human Superoxide dismutase 1 (hSOD1) gene are well‐established cause of the motor neuron disease ALS. Patients and transgenic (Tg) ALS model mice carrying mutant variants develop hSOD1 aggregates in the CNS. We have identified two hSOD1 aggregate strains, which both transmit spreading template‐directed aggregation and premature fatal paralysis when inoculated into adult transgenic mice. This prion‐like spread of aggregation could be a primary disease mechanism in SOD1‐induced ALS. Human SOD1 aggregation has been studied extensively both in cultured cells and under various conditions in vitro. To determine how the structure of aggregates formed in these model systems related to disease‐associated aggregates in the CNS, we used a binary epitope‐mapping assay to examine aggregates of hSOD1 variants G93A, G85R, A4V, D90A, and G127X formed in vitro, in four different cell lines and in the CNS of Tg mice. We found considerable variability between replicate sets of in vitro‐generated aggregates. In contrast, there was a high similarity between replicates of a given hSOD1 mutant in a given cell line, but pronounced variations between different hSOD1 mutants and different cell lines in both structures and amounts of aggregates formed. The aggregates formed in vitro or in cultured cells did not replicate the aggregate strains that arise in the CNS. Our findings suggest that the distinct aggregate morphologies in the CNS could result from a micro‐environment with stringent quality control combined with second‐order selection by spreading ability. Explorations of pathogenesis and development of therapeutics should be conducted in models that replicate aggregate structures forming in the CNS. image

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

Aggregate formation of superoxide dismutase 1 (SOD1) inside motor neurons is known as a major factor in onset of amyotrophic lateral sclerosis. The thermodynamic stability of the SOD1 β-barrel has been shown to decrease in crowded environments such as inside a cell, but it remains unclear how the thermodynamics of crowding-induced protein destabilization relate to SOD1 aggregation. Here we have examined the effects of a protein crowder, lysozyme, on fibril aggregate formation of the SOD1 β-barrel. We found that aggregate formation of SOD1 is decelerated even in mildly crowded solutions. Intriguingly, transient diffusive interactions with lysozyme do not significantly affect the static structure of the SOD1 β-barrel but stabilize an alternative excited “invisible” state. The net effect of crowding is to favor species off the aggregation pathway, thereby explaining the decelerated aggregation in the crowded environment. Our observations suggest that the intracellular environment may have a similar negative (inhibitory) effect on fibril formation of other amyloidogenic proteins in living cells. Deciphering how crowded intracellular environments affect aggregation and fibril formation of such disease-associated proteins will probably become central in understanding the exact role of aggregation in the etiology of these enigmatic diseases.

The structural stability of proteins is found to markedly change upon their transfer to the crowded interior of live cells. For some proteins, the stability increases, while for others, it decreases, depending on both the sequence composition and the type of host cell. The mechanism seems to be linked to the strength and conformational bias of the diffusive in-cell interactions, where protein charge is found to play a decisive role. Because most proteins, nucleotides, and membranes carry a net-negative charge, the intracellular environment behaves like a polyanionic (Z:1) system with electrostatic interactions different from those of standard 1:1 ion solutes. To determine how such polyanion conditions influence protein stability, we use negatively charged polyacetate ions to mimic the net-negatively charged cellular environment. The results show that, per Na⁺ equivalent, polyacetate destabilizes the model protein SOD1^barrel significantly more than monoacetate or NaCl. At an equivalent of 100 mM Na⁺, the polyacetate destabilization of SOD1^barrel is similar to that observed in live cells. By the combined use of equilibrium thermal denaturation, folding kinetics, and high-resolution nuclear magnetic resonance, this destabilization is primarily assigned to preferential interaction between polyacetate and the globally unfolded protein. This interaction is relatively weak and involves mainly the outermost N-terminal region of unfolded SOD1^barrel. Our findings point thus to a generic influence of polyanions on protein stability, which adds to the sequence-specific contributions and needs to be considered in the evaluation of in vivo data.

In the cytosolic environment, protein crowding and Brownian motions result in numerous transient encounters. Each such encounter event increases the apparent size of the interacting molecules, leading to slower rotational tumbling. The extent of transient protein complexes formed in live cells can conveniently be quantified by an apparent viscosity, based on NMR-detected spin-relaxation measurements, that is, the longitudinal (T₁) and transverse (T₂) relaxation. From combined analysis of three different proteins and surface mutations thereof, we find that T₂ implies significantly higher apparent viscosity than T₁. At first sight, the effect on T₁ and T₂ seems thus nonunifiable, consistent with previous reports on other proteins. We show here that the T₁ and T₂ deviation is actually not a inconsistency but an expected feature of a system with fast exchange between free monomers and transient complexes. In this case, the deviation is basically reconciled by a model with fast exchange between the free-tumbling reporter protein and a transient complex with a uniform 143 kDa partner. The analysis is then taken one step further by accounting for the fact that the cytosolic content is by no means uniform but comprises a wide range of molecular sizes. Integrating over the complete size distribution of the cytosolic interaction ensemble enables us to predict both T₁ and T₂ from a single binding model. The result yields a bound population for each protein variant and provides a quantification of the transient interactions. We finally extend the approach to obtain a correction term for the shape of a database-derived mass distribution of the interactome in the mammalian cytosol, in good accord with the existing data of the cellular composition.

Significance: Despite the vast number of known protein topologies, the actual behavior and selective traits of their constituent α- and β-secondary-structure elements remain elusive. The reason is that the high-energy fluctuations associated with their motions in folded proteins are intrinsically difficult to measure. Here, we circumvent this problem by presenting an alternative, and generally applicable, protein-design strategy that activates dynamic swapping of entire α- and β-elements in the folded ground state. We find that the behavior of the α- and β-elements is, indeed, very distinct where the differences in structural and dynamic properties have relevance not only for the understanding of protein function, but also for the mechanism of structural evolution per se.

In the disordered state, a protein exhibits a high degree of structural freedom, in both space and time. For an ensemble of disordered or unfolded proteins, this means that the ensemble comprises a high diversity of structures, ranging from compact collapsed states to fully extended polypeptide chains. In addition, each chain is highly dynamic and undergoes conformational changes and local dynamics on both fast and slow timescales. The size properties of disordered proteins are thus best described as ensemble averages. A straightforward measure of the size is the hydrodynamic radius, R_H, of the ensemble. Since the disordered state is conformationally fluid, the observed R_H does not refer to a particular shape or fold. Instead, it should be interpreted as a measure for the average compaction of the structural ensemble. In addition to characterizing the disordered ensemble itself, R_H can be used to, with good precision, monitor changes in the ensemble size properties upon functional interactions of the disordered protein, e.g., dimerization, ligand binding, and folding pathways. Here, we present a step-by-step protocol for diffusion measurements using pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. We describe how to calibrate the magnetic field gradient and offer different schemes for sample preparation. Finally, we describe how to obtain R_H directly from the diffusion coefficient as well as from using an internal standard as a reference.

Cellular function is generally depicted at the level of functional pathways and detailed structural mechanisms, based on the identification of specific protein–protein interactions. For an individual protein searching for its partner, however, the perspective is quite different: The functional task is challenged by a dense crowd of nonpartners obstructing the way. Adding to the challenge, there is little information about how to navigate the search, since the encountered surrounding is composed of protein surfaces that are predominantly “nonconserved” or, at least, highly variable across organisms. In this study, we demonstrate from a colloidal standpoint that such a blindfolded intracellular search is indeed favored and has more fundamental impact on the cellular organization than previously anticipated. Basically, the unique polyion composition of cellular systems renders the electrostatic interactions different from those in physiological buffer, leading to a situation where the protein net-charge density balances the attractive dispersion force and surface heterogeneity at close range. Inspection of naturally occurring proteomes and in-cell NMR data show further that the “nonconserved” protein surfaces are by no means passive but chemically biased to varying degree of net-negative repulsion across organisms. Finally, this electrostatic control explains how protein crowding is spontaneously maintained at a constant level through the intracellular osmotic pressure and leads to the prediction that the “extreme” in halophilic adaptation is not the ionic-liquid conditions per se but the evolutionary barrier of crossing its physicochemical boundaries.