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1. Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

   https://doi.org/10.3390/cryst11080874  

   Oestreicher, Zachery ; Valverde-Tercedor, Carmen ; Mumper, Eric ; Pérez-Guzmán, Lumarie ; Casillas-Ituarte, Nadia N. ; Jimenez-Lopez, Concepcion ; Bazylinski, Dennis A. ; Lower, Steven K. ; Lower, Brian H. ( August 2021 , Crystals)

   Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digestedmore »with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.« less
2. Electroblotting through a tryptic membrane for LC-MS/MS analysis of proteins separated in electrophoretic gels

   https://doi.org/10.1039/D0AN01380C  

   Bickner, A. N. ; Champion, M. M. ; Hummon, A. B. ; Bruening, M. L. ( November 2020 , The Analyst)

   Digestion of proteins separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) remains a popular method for protein identification using mass-spectrometry based proteomics. Although robust and routine, the in-gel digestion procedure is laborious and time-consuming. Electroblotting to a capture membrane prior to digestion reduces preparation steps but requires on-membrane digestion that yields fewer peptides than in-gel digestion. This paper develops direct electroblotting through a trypsin-containing membrane to a capture membrane to simplify extraction and digestion of proteins separated by SDS-PAGE. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies the extracted peptides. Analysis of peptides from different capture membrane pieces shows that electrodigestion does not greatly disturb the spatial resolution of a standard protein mixture separated by SDS-PAGE. Electrodigestion of an Escherichia coli ( E. coli ) cell lysate requires four hours of total sample preparation and results in only 13% fewer protein identifications than in-gel digestion, which can take 24 h. Compared to simple electroblotting and protein digestion on a poly(vinylidene difluoride) (PVDF) capture membrane, adding a trypsin membrane to the electroblot increases the number of protein identifications by 22%. Additionally, electrodigestion experiments using capture membranes coated with polyelectrolyte layers identify a higher fraction of small proteolytic peptides than capture onmore »PVDF or in-gel digestion.« less
3. Segmented flow generator for serial crystallography at the European X-ray free electron laser

   https://doi.org/10.1038/s41467-020-18156-7  

   Echelmeier, Austin ; Cruz Villarreal, Jorvani ; Messerschmidt, Marc ; Kim, Daihyun ; Coe, Jesse D. ; Thifault, Darren ; Botha, Sabine ; Egatz-Gomez, Ana ; Gandhi, Sahir ; Brehm, Gerrit ; et al ( September 2020 , Nature Communications)

   Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
4. Effects of amino acid additives on protein solubility – insights from desorption and direct electrospray ionization mass spectrometry

   https://doi.org/10.1039/D1AN01392K  

   Javanshad, Roshan ; Venter, Andre R. ( October 2021 , The Analyst)

   Naturally occurring amino acids have been broadly used as additives to improve protein solubility and inhibit aggregation. In this study, improvements in protein signal intensity obtained with the addition of l -serine, and structural analogs, to the desorption electrospray ionization mass spectrometry (DESI-MS) spray solvent were measured. The results were interpreted at the hand of proposed mechanisms of solution additive effects on protein solubility and dissolution. DESI-MS allows for these processes to be studied efficiently using dilute concentrations of additives and small amounts of proteins, advantages that represent real benefits compared to classical methods of studying protein stability and aggregation. We show that serine significantly increases the protein signal in DESI-MS when native proteins are undergoing unfolding during the dissolution process with an acidic solvent system ( p -value = 0.0001), or with ammonium bicarbonate under denaturing conditions for proteins with high isoelectric points ( p -value = 0.001). We establish that a similar increase in the protein signal cannot be observed with direct ESI-MS, and the observed increase is therefore not related to ionization processes or changes in the physical properties of the bulk solution. The importance of the presence of serine during protein conformational changes while undergoing dissolutionmore »is demonstrated through comparisons between the analyses of proteins deposited in native or unfolded states and by using native state-preserving and denaturing desorption solvents. We hypothesize that direct, non-covalent interactions involving all three functional groups of serine are involved in the beneficial effect on protein solubility and dissolution. Supporting evidence for a direct interaction include a reduction in efficacy with d -serine or the racemic mixture, indicating a non-bulk-solution physical property effect; insensitivity to the sample surface type or relative placement of serine addition; and a reduction in efficacy with any modifications to the serine structure, most notably the carboxyl functional group. An alternative hypothesis, also supported by some of our observations, could involve the role of serine clusters in the mechanism of solubility enhancement. Our study demonstrates the capability of DESI-MS together with complementary ESI-MS experiments as a novel tool for understanding protein solubility and dissolution and investigating the mechanism of action for solubility-enhancing additives.« less
5. SVM-SulfoSite: A support vector machine based predictor for sulfenylation sites

   https://doi.org/10.1038/s41598-018-29126-x  

   AL-barakati, Hussam J. ; McConnell, Evan W. ; Hicks, Leslie M. ; Poole, Leslie B. ; Newman, Robert H. ; KC, Dukka B. ( July 2018 , Scientific Reports)

   ProteinS-sulfenylation, which results from oxidation of free thiols on cysteine residues, has recently emerged as an important post-translational modification that regulates the structure and function of proteins involved in a variety of physiological and pathological processes. By altering the size and physiochemical properties of modified cysteine residues, sulfenylation can impact the cellular function of proteins in several different ways. Thus, the ability to rapidly and accurately identify putative sulfenylation sites in proteins will provide important insights into redox-dependent regulation of protein function in a variety of cellular contexts. Though bottom-up proteomic approaches, such as tandem mass spectrometry (MS/MS), provide a wealth of information about global changes in the sulfenylation state of proteins, MS/MS-based experiments are often labor-intensive, costly and technically challenging. Therefore, to complement existing proteomic approaches, researchers have developed a series of computational tools to identify putative sulfenylation sites on proteins. However, existing methods often suffer from low accuracy, specificity, and/or sensitivity. In this study, we developed SVM-SulfoSite, a novel sulfenylation prediction tool that uses support vector machines (SVM) to identify key determinants of sulfenylation among five feature classes: binary code, physiochemical properties, k-space amino acid pairs, amino acid composition and high-quality physiochemical indices. Using 10-fold cross-validation, SVM-SulfoSite achievedmore »95% sensitivity and 83% specificity, with an overall accuracy of 89% and Matthew’s correlation coefficient (MCC) of 0.79. Likewise, using an independent test set of experimentally identified sulfenylation sites, our method achieved scores of 74%, 62%, 80% and 0.42 for accuracy, sensitivity, specificity and MCC, with an area under the receiver operator characteristic (ROC) curve of 0.81. Moreover, in side-by-side comparisons, SVM-SulfoSite performed as well as or better than existing sulfenylation prediction tools. Together, these results suggest that our method represents a robust and complementary technique for advanced exploration of protein S-sulfenylation.

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