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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., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

In structural biology, collision cross sections (CCSs) from ion mobility mass spectrometry (IM-MS) measurements are routinely compared to computationally or experimentally derived protein structures. Here, we investigate whether CCS data can inform about the shape of a protein in the absence of specific reference structures. Analysis of the proteins in the CCS database shows that protein complexes with low apparent densities are structurally more diverse than those with a high apparent density. Although assigning protein shapes purely on CCS data is not possible, we find that we can distinguish oblate- and prolate-shaped protein complexes by using the CCS, molecular weight, and oligomeric states to mine the Protein Data Bank (PDB) for potentially similar protein structures. Furthermore, comparing the CCS of a ferritin cage to the solution structures in the PDB reveals significant deviations caused by structural collapse in the gas phase. We then apply the strategy to an integral membrane protein by comparing the shapes of a prokaryotic and a eukaryotic sodium/proton antiporter homologue. We conclude that mining the PDB with IM-MS data is a time-effective way to derive low-resolution structural models.

Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic-to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.

Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine- N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine- N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.

Ion mobility mass spectrometry of integral membrane proteins provides valuable insights into their architecture and stability. Here we show that, due to their lower charge, the average mobility of native-like membrane protein ions is approximately 30% lower than that of soluble proteins of similar mass. This has implications for drift time measurements, made on traveling wave ion mobility mass spectrometers, which have to be calibrated to extract collision cross sections (Ω). Common calibration strategies employ unfolded or native-like soluble protein standards with masses and mobilities comparable to the protein of interest. We compare Ω values for membrane proteins, derived from standard calibration protocols using soluble proteins, to values measured using an RF-confined drift tube. Our results demonstrate that, while common calibration methods underestimate Ω for native-like or unfolded membrane protein complexes, higher mass soluble calibration standards consistently yield more accurate Ω values. These findings enable us to obtain directly structural information for highly charge-reduced complexes by traveling wave ion mobility mass spectrometry., Competing Interests: Notes The authors declare no competing financial interest.

The study of intact soluble protein assemblies by means of mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar study of membrane protein complexes, following their release from detergent micelles in the gas phase. Careful optimization of mass spectrometry conditions, particularly with respect to energy regimes, is essential for maintaining compact folded states as detergent is removed. However, many of the saccharide detergents widely employed in structural biology can cause unfolding of membrane proteins in the gas phase. Here, we investigate the potential of charge reduction by introducing three membrane protein complexes from saccharide detergents and show how reducing their overall charge enables generation of compact states, as evidenced by ion mobility mass spectrometry. We find that charge reduction stabilizes the oligomeric state and enhances the stability of lipid-bound complexes. This finding is significant since maintaining native-like membrane proteins enables ligand binding to be assessed from a range of detergents that retain solubility while protecting the overall fold.

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the pathway to 3-deoxy-d-manno-octulosonate, a component in the cell wall of Gram-negative bacteria. KDO8PS is evolutionarily and structurally related to the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), which uses erythrose 4-phosphate in place of A5P. Both KDO8PS and type Iβ DAH7PS enzymes adopt similar homotetrameric associations with their active sites close to one of the interfaces. The conserved PAFLxR motif in KDO8PS and the corresponding GARNxQ motif in type Iβ DAH7PS, both on the short β4-α4 loop of the (β/α)8 barrel, form part of this interface and provide key contacts with substrates. This (112)PAFLxR(117) motif was mutated in Neisseria meningitidis KDO8PS in order to assess its role in enzyme function. Arg117 extends across the interface to provide guanidinium functionality in the A5P binding site of the adjacent subunit. Substitution Arg117Ala severely hampered catalysis, whereas substitution to Lys was tolerated better. Mutation of Phe114 to either Arg or Ala results in active proteins, but with substantially elevated Km(A5P) values. Mutant proteins that combine substitutions in this motif demonstrate poor catalytic function, and, although these mutated residues now structurally resemble their counterparts in the GARNxQ motif of type Iβ DAH7PS, no DAH7PS-like activity was observed. Analysis of the structures reveals that small changes in relative orientation of the subunits are important for the differences in active-site construction. Quaternary structure is therefore tightly linked to substrate specificity.

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the biosynthetic pathway for the formation of 3-deoxy-D-manno-octulosonate, an important component in the cell wall of Gram-negative bacteria. KDO8P synthase is evolutionarily related to the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase, which uses erythrose 4-phosphate in place of A5P. The A5P binding site in KDO8P synthase is formed by three long loops that extend from the core catalytic (β/α)(8) barrel, β2α2, β7α7, and β8α8. The extended β7α7 loop is always present in KDO8P synthase yet is not observed for DAH7P synthase. Modeling of this loop indicated interactions between this loop and the extended β2α2 loop; both loops provide key hydrogen-bonding contacts with A5P. The two absolutely conserved residues on the β7α7 loop (Gln and Ser) were mutated to Ala in both the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans and the metal-independent KDO8P synthase from Neisseria meningitidis. In addition, mutants were constructed for both enzymes with the extended β7α7 loop excised to match the DAH7P synthase architecture. Removal of the loop extension severely hindered efficient catalysis, dramatically increasing the K(m)(A5P) and reducing the k(cat) for both enzymes. Excision of the complete loop was far more detrimental to catalysis than the double mutations of the two conserved Gln and Ser residues. Therefore, the presence of the entire extended β7α7 loop is important for efficient catalysis by KDO8P synthase, with the loop acting to promote efficient and productive binding of A5P.

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.