Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles.

Collision induced unfolding (CIU) is a method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the gas-phase unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation, together with CIU experiments. The dimers have highly similar structures, but their CIU reveals different stability that can be explained by the different dynamics that arises in response to the activation seen in the simulations, including a part of the sequence with previously observed strain-specific dynamics in solution. Our findings show how similar protein variants can be examined using mass spectrometric techniques in conjunction with atomistic molecular dynamics simulations to reveal differences in stability as well as differences in how and where unfolding takes place upon activation.

Rehydration Post-orientation: Investigating Field-Induced Structural Changes via Computational Rehydration.

Proteins can be oriented in the gas phase using strong electric fields, which brings advantages for structure determination using X-ray free electron lasers. Both the vacuum conditions and the electric-field exposure risk damaging the protein structures. Here, we employ molecular dynamics simulations to rehydrate and relax vacuum and electric-field exposed proteins in aqueous solution, which simulates a refinement of structure models derived from oriented gas-phase proteins. We find that the impact of the strong electric fields on the protein structures is of minor importance after rehydration, compared to that of vacuum exposure and ionization in electrospraying. The structures did not fully relax back to their native structure in solution on the simulated timescales of 200 ns, but they recover several features, including native-like intra-protein contacts, which suggests that the structures remain in a state from which the fully native structure is accessible. Our findings imply that the electric fields used in native mass spectrometry are well below a destructive level, and suggest that structures inferred from X-ray diffraction from gas-phase proteins are relevant for solution and in vivo conditions, at least after in silico rehydration.

Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

Stability and conformational memory of electrosprayed and rehydrated bacteriophage MS2 virus coat proteins.

Proteins are innately dynamic, which is important for their functions, but which also poses significant challenges when studying their structures. Gas-phase techniques can utilise separation and a range of sample manipulations to transcend some of the limitations of conventional techniques for structural biology in crystalline or solution phase, and isolate different states for separate interrogation. However, the transfer from solution to the gas phase risks affecting the structures, and it is unclear to what extent different conformations remain distinct in the gas phase, and if resolution in silico can recover the native conformations and their differences. Here, we use extensive molecular dynamics simulations to study the two distinct conformations of dimeric capsid protein of the MS2 bacteriophage. The protein undergoes notable restructuring of its peripheral parts in the gas phase, but subsequent simulation in solvent largely recovers the native structure. Our results suggest that despite some structural loss due to the experimental conditions, gas-phase structural biology techniques provide meaningful data that inform not only about the structures but also conformational dynamics of proteins.

Glycan-Induced Protein Dynamics in Human Norovirus P Dimers Depend on Virus Strain and Deamidation Status.

Noroviruses are the major cause of viral gastroenteritis and re-emerge worldwide every year, with GII.4 currently being the most frequent human genotype. The norovirus capsid protein VP1 is essential for host immune response. The P domain mediates cell attachment via histo blood-group antigens (HBGAs) in a strain-dependent manner but how these glycan-interactions actually relate to cell entry remains unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is used to investigate glycan-induced protein dynamics in P dimers of different strains, which exhibit high structural similarity but different prevalence in humans. While the almost identical strains GII.4 Saga and GII.4 MI001 share glycan-induced dynamics, the dynamics differ in the emerging GII.17 Kawasaki 308 and rare GII.10 Vietnam 026 strain. The structural aspects of glycan binding to fully deamidated GII.4 P dimers have been investigated before. However, considering the high specificity and half-life of N373D under physiological conditions, large fractions of partially deamidated virions with potentially altered dynamics in their P domains are likely to occur. Therefore, we also examined glycan binding to partially deamidated GII.4 Saga and GII.4 MI001 P dimers. Such mixed species exhibit increased exposure to solvent in the P dimer upon glycan binding as opposed to pure wildtype. Furthermore, deamidated P dimers display increased flexibility and a monomeric subpopulation. Our results indicate that glycan binding induces strain-dependent structural dynamics, which are further altered by N373 deamidation, and hence hint at a complex role of deamidation in modulating glycan-mediated cell attachment in GII.4 strains.

Structural Heterogeneity in Single Particle Imaging Using X-ray Lasers.

One of the challenges facing single particle imaging with ultrafast X-ray pulses is the structural heterogeneity of the sample to be imaged. For the method to succeed with weakly scattering samples, the diffracted images from a large number of individual proteins need to be averaged. The more the individual proteins differ in structure, the lower the achievable resolution in the final reconstructed image. We use molecular dynamics to simulate two globular proteins in vacuum, fully desolvated as well as with two different solvation layers, at various temperatures. We calculate the diffraction patterns based on the simulations and evaluate the noise in the averaged patterns arising from the structural differences and the surrounding water. Our simulations show that the presence of a minimal water coverage with an average 3 Å thickness will stabilize the protein, reducing the noise associated with structural heterogeneity, whereas additional water will generate more background noise.

Reproducibility in the unfolding process of protein induced by an external electric field.

The dynamics of proteins are crucial for their function. However, commonly used techniques for studying protein structures are limited in monitoring time-resolved dynamics at high resolution. Combining electric fields with existing techniques to study gas-phase proteins, such as single particle imaging using free-electron lasers and gas-phase small angle X-ray scattering, has the potential to open up a new era in time-resolved studies of gas-phase protein dynamics. Using molecular dynamics simulations, we identify well-defined unfolding pathways of a protein, induced by experimentally achievable external electric fields. Our simulations show that strong electric fields in conjunction with short-pulsed X-ray sources such as free-electron lasers can be a new path for imaging dynamics of gas-phase proteins at high spatial and temporal resolution.