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1. Integrating CORUM and co-fractionation mass spectrometry to create enhanced benchmarks for protein complex predictions

   https://doi.org/10.1093/bib/bbaf154  

   Yang, Pengcheng; Lee, Youngwoo; Szymanski, Daniel B; Xie, Jun (March 2025, Briefings in Bioinformatics)

   Abstract Co-fractionation mass spectrometry (CFMS) enables the discovery of protein complexes and the systems-level analysis of multimer dynamics that facilitate responses to environmental and developmental conditions. A major challenge in CFMS data analysis, and omics approaches in general, is the development of reliable benchmarks for accurate evaluation of prediction methods. CORUM is commonly used as a source of benchmark complexes for protein complex composition predictions; however, its assumption of fully assembled subunit pools often conflicts with size exclusion chromatography (SEC) and interaction predictions from CFMS experiments. To address this, we developed an integrative analysis method that leverages cross-kingdom evolutionary conservation among specific CORUM complexes and high-resolution SEC profile data from cell extracts. The resulting benchmark complexes are supported by statistical significance and consistent sizes between calculated and measured apparent masses. The approach was robust, revealing both conserved and species-specific complexes. Designed specifically for benchmark identification, this method can be applied to any species and used to evaluate protein complex predictions from other studies. 

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2. Structural and dynamic studies of the human RNA binding protein RBM3 reveals the molecular basis of its oligomerization and RNA recognition

   https://doi.org/10.1111/febs.16301  

   Roy, Sayantani; Boral, Soumendu; Maiti, Snigdha; Kushwaha, Tushar; Basak, Aditya J.; Lee, Woonghee; Basak, Amit; Gholap, Shivajirao L.; Inampudi, Krishna K.; De, Soumya (December 2021, The FEBS Journal)

   Human RNA‐binding motif 3 protein (RBM3) is a cold‐shock protein which functions in various aspects of global protein synthesis, cell proliferation and apoptosis by interacting with the components of basal translational machinery. RBM3 plays important roles in tumour progression and cancer metastasis, and also has been shown to be involved in neuroprotection and endoplasmic reticulum stress response. Here, we have solved the solution NMR structure of the N‐terminal 84 residue RNA recognition motif (RRM) of RBM3. The remaining residues are rich in RGG and YGG motifs and are disordered. The RRM domain adopts a βαββαβ topology, which is found in many RNA‐binding proteins. NMR‐monitored titration experiments and molecular dynamic simulations show that the beta‐sheet and two loops form the RNA‐binding interface. Hydrogen bond, pi–pi and pi–cation are the key interactions between the RNA and the RRM domain. NMR, size exclusion chromatography and chemical cross‐linking experiments show that RBM3 forms oligomers in solution, which is favoured by decrease in temperature, thus, potentially linking it to its function as a cold‐shock protein. Temperature‐dependent NMR studies revealed that oligomerization of the RRM domain occurs via nonspecific interactions. Overall, this study provides the detailed structural analysis of RRM domain of RBM3, its interaction with RNA and the molecular basis of its temperature‐dependent oligomerization. 

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3. Allosteric regulation in SARS-CoV-2 spike protein

   https://doi.org/10.1039/D4CP00106K  

   Wei, Yong; Chen, Amy X; Lin, Yuewei; Wei, Tao; Qiao, Baofu (February 2024, Physical Chemistry Chemical Physics)

   Allosteric regulation is common in protein–protein interactions and is thus promising in drug design. Previous experimental and simulation work supported the presence of allosteric regulation in the SARS-CoV-2 spike protein. Here the route of allosteric regulation in SARS-CoV-2 spike protein is examined by all-atom explicit solvent molecular dynamics simulations, contrastive machine learning, and the Ohm approach. It was found that peptide binding to the polybasic cleavage sites, especially the one at the first subunit of the trimeric spike protein, activates the fluctuation of the spike protein's backbone, which eventually propagates to the receptor-binding domain on the third subunit that binds to ACE2. Remarkably, the allosteric regulation routes starting from the polybasic cleavage sites share a high fraction (39–67%) of the critical amino acids with the routes starting from the nitrogen-terminal domains, suggesting the presence of an allosteric regulation network in the spike protein. Our study paves the way for the rational design of allosteric antibody inhibitors. 

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4. SurA is a cryptically grooved chaperone that expands unfolded outer membrane proteins

   https://doi.org/10.1073/pnas.2008175117  

   Marx, Dagan C.; Plummer, Ashlee M.; Faustino, Anneliese M.; Devlin, Taylor; Roskopf, Michaela A.; Leblanc, Mathis J.; Lessen, Henry J.; Amann, Barbara T.; Fleming, Patrick J.; Krueger, Susan; et al (October 2020, Proceedings of the National Academy of Sciences)

   The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the β-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network. 

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5. Tandem trapped ion mobility spectrometry

   https://doi.org/10.1039/c7an02054f  

   Liu, Fanny C.; Ridgeway, Mark E.; Park, Melvin A.; Bleiholder, Christian (January 2018, The Analyst)

   There is currently a strong interest in the use of ion mobility spectrometry-mass spectrometry (IMS-MS) instrumentation for structural biology. In these applications, momentum transfer cross sections derived from IMS-MS measurements are used to reconstruct the three-dimensional analyte structure. Recent reports indicate that additional structural information can be extracted from measuring changes in cross sections in response to changes of the analyte structure. To further this approach, we constructed a tandem trapped IMS analyser (TIMS-TIMS) and incorporated it in a QqTOF mass spectrometer. TIMS-TIMS is constructed by coupling two TIMS analysers via an “interface region” composed of two apertures. We show that peptide oligomers (bradykinin) and native-like protein (ubiquitin) ions can be preserved through the course of an experiment in a TIMS-TIMS analyser. We demonstrate the ability to collisionally-activate as well as to trap mobility-selected ions, followed by subsequent mobility-analysis. In addition to inducing conformational changes, we show that we can fragment low charge states of ubiquitin at >1 mbar between the TIMS analysers with significant sequence coverage. Many fragment ions exhibit multiple features in their TIMS spectra, which means that they may not generally exist as the most stable isomer. The ability of TIMS-TIMS to dissociate mobility-selected protein ions and to measure the cross sections of their fragment ions opens new possibilities for IMS-based structure elucidation. 

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