Cold Denaturation of Proteins in the Absence of Solvent: Implications for Protein Storage.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Cold Denaturation of Proteins in the Absence of Solvent: Implications for Protein Storage.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

Deuteros 2.0: peptide-level significance testing of data from hydrogen deuterium exchange mass spectrometry.

SUMMARY: Hydrogen deuterium exchange mass spectrometry (HDX-MS) is becoming increasing routine for monitoring changes in the structural dynamics of proteins. Differential HDX-MS allows comparison of protein states, such as in the absence or presence of a ligand. This can be used to attribute changes in conformation to binding events, allowing the mapping of entire conformational networks. As such, the number of necessary cross-state comparisons quickly increases as additional states are introduced to the system of study. There are currently very few software packages available that offer quick and informative comparison of HDX-MS datasets and even fewer which offer statistical analysis and advanced visualization. Following the feedback from our original software Deuteros, we present Deuteros 2.0 which has been redesigned from the ground up to fulfill a greater role in the HDX-MS analysis pipeline. Deuteros 2.0 features a repertoire of facilities for back exchange correction, data summarization, peptide-level statistical analysis and advanced data plotting features. AVAILABILITY AND IMPLEMENTATION: Deuteros 2.0 can be downloaded for both Windows and MacOS from https://github.com/andymlau/Deuteros_2.0 under the Apache 2.0 license.

An Unusually Rapid Protein Backbone Modification Stabilizes the Essential Bacterial Enzyme MurA.

Proteins are subject to spontaneous rearrangements of their backbones. Most prominently, asparagine and aspartate residues isomerize to their ß-linked isomer, isoaspartate (isoAsp), on time scales ranging from days to centuries. Such modifications are typically considered "molecular wear-and-tear", destroying protein function. However, the observation that some proteins, including the essential bacterial enzyme MurA, harbor stoichiometric amounts of isoAsp suggests that this modification can confer advantageous properties. Here, we demonstrate that nature exploits an isoAsp residue within a hairpin to stabilize MurA. We found that isoAsp formation in MurA is unusually rapid and critically dependent on folding status. Moreover, perturbation of the isoAsp-containing hairpin via site-directed mutagenesis causes aggregation of MurA variants. Structural mass spectrometry revealed that this effect is caused by local protein unfolding in MurA mutants. Our findings demonstrate that MurA evolved to "mature" via a spontaneous post-translational incorporation of a ß-amino acid, which raises the possibility that isoAsp-containing hairpins may serve as a structural motif of biological importance.

Improving Peptide Fragmentation for Hydrogen-Deuterium Exchange Mass Spectrometry Using a Time-Dependent Collision Energy Calculator.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is becoming a popular technique for interrogating biological systems. In recent years, advancements have been made to increase peptide coverage for proteins that resist digestion such as antibodies and membrane proteins. These methods commonly include using alternative digestion enzymes or longer chromatographic gradients, which may be expensive or time-consuming to implement. Here, we recommend an efficient proteomics-based approach to increase peptide confidence and coverage. A major filtering parameter for peptides in HDX is the number of product ions detected; this is a result of the collision energy (CE) applied within the MS. A traditional linear ramp achieves optimal CE for only short periods of time. More product ions will be created and detected if optimal CE can be achieved for a longer period of time. As a result, the coverage, redundancy, and data confidence are all increased. We achieved this by implementing a mobility-dependent CE look up table (LUT) which increases the CE as a function of mobility. We developed a program to calculate the optimal CE for a set of peptides and MS settings based on initial reference samples. We demonstrated the utility of the CE LUT on three protein samples including the soluble phosphorylase B, IgG2, and the membrane-stabilized AcrB. We showed that applying a CE LUT provided 8.5-50% more peptides compared to a linear CE ramp. The results demonstrate that a time-dependent CE LUT is a quick and inexpensive method to increase data confidence and peptide abundance for HDX-MS experiments.

Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome.

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

A Mass-Spectrometry-Based Modelling Workflow for Accurate Prediction of IgG Antibody Conformations in the Gas Phase.

Immunoglobulins are biomolecules involved in defence against foreign substances. Flexibility is key to their functional properties in relation to antigen binding and receptor interactions. We have developed an integrative strategy combining ion mobility mass spectrometry (IM-MS) with molecular modelling to study the conformational dynamics of human IgG antibodies. Predictive models of all four human IgG subclasses were assembled and their dynamics sampled in the transition from extended to collapsed state during IM-MS. Our data imply that this collapse of IgG antibodies is related to their intrinsic structural features, including Fab arm flexibility, collapse towards the Fc region, and the length of their hinge regions. The workflow presented here provides an accurate structural representation in good agreement with the observed collision cross section for these flexible IgG molecules. These results have implications for studying other nonglobular flexible proteins.

Structural basis for isoform-specific kinesin-1 recognition of Y-acidic cargo adaptors.

The light chains (KLCs) of the heterotetrameric microtubule motor kinesin-1, that bind to cargo adaptor proteins and regulate its activity, have a capacity to recognize short peptides via their tetratricopeptide repeat domains (KLCTPR). Here, using X-ray crystallography, we show how kinesin-1 recognizes a novel class of adaptor motifs that we call 'Y-acidic' (tyrosine flanked by acidic residues), in a KLC-isoform-specific manner. Binding specificities of Y-acidic motifs (present in JIP1 and in TorsinA) to KLC1TPR are distinct from those utilized for the recognition of W-acidic motifs, found in adaptors, that are KLC-isoform non-selective. However, a partial overlap on their receptor-binding sites implies that adaptors relying on Y-acidic and W-acidic motifs must act independently. We propose a model to explain why these two classes of motifs that bind to the concave surface of KLCTPR with similar low micromolar affinity can exhibit different capacities to promote kinesin-1 activity.

Glu-C, an alternative digestive enzyme for the quantitative LC-MS/MS analysis of an IgG-based antibody biotherapeutic.

AIM: LC-MS/MS bottom-up quantitation of proteins has become increasingly popular with trypsin as the most commonly used protease. However, trypsin does not always yield suitable surrogate peptides. An alternative enzyme, Glu-C, was used to generate surrogate peptides for quantifying a bispecific IgG1 biotherapeutic antibody in preclinical matrices.  Materials and methods: IgG1 was quantified by pellet digestion using an Acquity UPLC coupled  with a Xevo TQ-S mass spectrometer.  Results: Two generic LC-MS/MS methods (heavy and light chain) were developed which afforded acceptable precision and accuracy, and an lower limit of quantitation of 1 µg/ml in three preclinical matrices. A small nonsignificant bias was observed when cynomolgus serum LC-MS/MS results were compared with electrochemiluminescent immunoassay data. CONCLUSION: Glu-C was successfully used as an alternative digestion enzyme for bottom-up quantitation of an IgG1 in matrices from multiple preclinical species, with good agreement with electrochemiluminescent immunoassay data.