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Alexander Tischer, Venkata R Machha, Juan P Frontroth, Maria A Brehm, Tobias Obser, Reinhard Schneppenheim, Leland Mayne, S Walter Englander, Matthew Auton
Mutation of the cysteines forming the disulfide loop of the platelet GPIbα adhesive A1 domain of von Willebrand factor (VWF) causes quantitative VWF deficiencies in the blood and von Willebrand disease. We report two cases of transient severe thrombocytopenia induced by DDAVP treatment. Cys1272Trp and Cys1458Tyr mutations identified by genetic sequencing implicate an abnormal gain-of-function phenotype, evidenced by thrombocytopenia, which quickly relapses back to normal platelet counts and deficient plasma VWF...
July 7, 2017: Journal of Molecular Biology
Patricia Aguilar-Calvo, Xiangzhu Xiao, Cyrus Bett, Hasier Eraña, Katrin Soldau, Joaquin Castilla, K Peter R Nilsson, Witold K Surewicz, Christina J Sigurdson
Misfolded prion protein aggregates (PrP(Sc)) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-β, tau, α-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)-anchorless prion protein, PrP(C), together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how post-translational modifications impact the aggregated prion protein properties and disease phenotype...
March 8, 2017: Scientific Reports
Geoffrey A Heinzl, Weiliang Huang, Wenbo Yu, Bennett J Giardina, Yue Zhou, Alexander D MacKerell, Angela Wilks, Fengtian Xue
New therapeutic targets are required to combat multidrug resistant infections, such as the iron-regulated heme oxygenase (HemO) of Pseudomonas aeruginosa, due to links between iron and virulence and dependence on heme as an iron source during infection. Herein we report the synthesis and activity of a series of iminoguanidine-based inhibitors of HemO. Compound 23 showed a binding affinity of 5.7 μM and an MIC50 of 52.3 μg/mL against P. aeruginosa PAO1. An in cellulo activity assay was developed by coupling HemO activity to a biliverdin-IXα-dependent infrared fluorescent protein, in which compound 23 showed an EC50 of 11...
July 28, 2016: Journal of Medicinal Chemistry
Jennine M Dawicki-McKenna, Marie-France Langelier, Jamie E DeNizio, Amanda A Riccio, Connie D Cao, Kelly R Karch, Michael McCauley, Jamin D Steffen, Ben E Black, John M Pascal
Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD(+) to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break...
December 3, 2015: Molecular Cell
Jacob Witten, Amy Ruschak, Timothy Poterba, Alexis Jaramillo, Andrew D Miranker, Sheila S Jaswal
The thermodynamic stability and kinetic barriers separating protein conformations under native conditions are critical for proper protein function and for understanding dysfunction in diseases of protein conformation. Traditional methods to probe protein unfolding and folding employ denaturants and highly non-native conditions, which may destabilize intermediate species or cause irreversible aggregation, especially at the high protein concentrations typically required. Hydrogen exchange (HX) is ideal for detecting conformational behavior under native conditions without the need for denaturants, but detection by NMR is limited to small highly soluble proteins...
August 6, 2015: Journal of Physical Chemistry. B
Minna Groenning, Raul I Campos, Daniel Hirschberg, Per Hammarström, Bente Vestergaard
Despite numerous studies, a detailed description of the transthyretin (TTR) self-assembly mechanism and fibril structure in TTR amyloidoses remains unresolved. Here, using a combination of primarily small -angle X-ray scattering (SAXS) and hydrogen exchange mass spectrometry (HXMS) analysis, we describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher average solvent exposure of TTR upon fibrillation...
2015: Scientific Reports
Adrian M Gospodarek, Weitong Sun, John P O'Connell, Erik J Fernandez
In hydrophobic interaction chromatography (HIC), interactions between buried hydrophobic residues and HIC surfaces can cause conformational changes that interfere with separations and cause yield losses. This paper extends our previous investigations of protein unfolding in HIC chromatography by identifying protein structures on HIC surfaces under denaturing conditions and relating them to solution behavior. The thermal unfolding of three model multidomain proteins on three HIC surfaces of differing hydrophobicities was investigated with hydrogen exchange mass spectrometry (HXMS)...
December 5, 2014: Journal of Chromatography. A
Victoria A Hughes, Robert Meklemburg, Stephen P Bottomley, Patrick L Wintrode
α1-Antitrypsin (α1AT) deficiency, the most common serpinopathy, results in both emphysema and liver disease. Over 90% of all clinical cases of α1AT deficiency are caused by the Z variant in which Glu342, located at the top of s5A, is replaced by a Lys which results in polymerization both in vivo and in vitro. The Glu342Lys mutation removes a salt bridge and a hydrogen bond but does not effect the thermodynamic stability of Z α1AT compared to the wild type protein, M α1AT, and so it is unclear why Z α1AT has an increased polymerization propensity...
2014: PloS One
Deepa Balasubramaniam, Elizabeth A Komives
Using HXMS to measure protein dynamics, Fang and colleagues (in this issue of Structure) probe the dynamics of processivity clamp proteins from all kingdoms of life. The proteins have similar structures but divergent sequences. Their results show conserved dynamics correlating with primary functions and divergent dynamics for divergent functions.
April 8, 2014: Structure
Alan Kadek, Hynek Mrazek, Petr Halada, Martial Rey, David C Schriemer, Petr Man
Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) utilizes enzymatic digestion of proteins to localize the information about altered exchange patterns in protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. Recently, it was shown that there is an interesting group of proteolytic enzymes from carnivorous pitcher plants of the genus Nepenthes. In this report, we describe successful immobilization and the use of one of these enzymes, nepenthesin-1, in HXMS workflow...
May 6, 2014: Analytical Chemistry
Sheila S Jaswal
Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes...
June 2013: Biochimica et Biophysica Acta
Deepa Balasubramaniam, Elizabeth A Komives
Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation...
June 2013: Biochimica et Biophysica Acta
Carsten Scavenius, Shirin Ghodke, Daniel E Otzen, Jan J Enghild
In this study, we have used glucagon as a model system for analyzing amyloid fibrillogenesis by hydrogen exchange MALDI mass spectrometry (HXMS). The hydrogen exchange mass spectrometry data correlated well with the traditional method based on Thioflavin T fluorescence and provided quantitative information by measuring the fibrillating molecules directly. The hydrogen exchange mass spectrometry data collected during fibrillogenesis revealed that glucagon fibrillation was a two component system showing an on/off type of interaction where only monomeric and fibrils were present without any substantial amount of intermediate species...
April 30, 2011: International Journal of Mass Spectrometry
James M Seckler, Mary D Barkley, Patrick L Wintrode
Efavirenz is a second-generation nonnucleoside reverse transcriptase inhibitor (NNRTI) and a common component of clinically approved anti-AIDS regimens. NNRTIs are noncompetitive inhibitors that bind in a hydrophobic pocket in the p66 subunit of reverse transcriptase (RT) ∼10 Å from the polymerase active site. Hydrogen exchange mass spectrometry (HXMS) shows that efavirenz binding reduces molecular flexibility in multiple regions of RT heterodimer in addition to the NNRTI binding site. Of the 47 peptic fragments monitored by HXMS, 15 showed significantly altered H/D exchange rates in the presence of efavirenz...
January 5, 2011: Biophysical Journal
Adrian M Gospodarek, Marissa E Smatlak, John P O'Connell, Erik J Fernandez
Hydrogen exchange mass spectrometry (HXMS) coupled to proteolytic digestion has been used to probe the conformation of bovine β-lactoglobulin (BLG), bovine α-lactalbumin (BLA), and human serum albumin (HSA) in solution and while adsorbed to the hydrophobic interaction chromatography media Phenyl Sepharose 6FF. All three proteins show evidence of EX1 exchange kinetics, indicating a loss of stability on the surface. HX protection patterns for all three proteins also indicate that the unfolded form is only partially solvent exposed...
January 4, 2011: Langmuir: the ACS Journal of Surfaces and Colloids
Valerie A Braz, Mary D Barkley, Rebecca A Jockusch, Patrick L Wintrode
Efavirenz (EFV) is a potent nonnucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of AIDS. NNRTIs bind in a hydrophobic pocket located in the p66 subunit of reverse transcriptase (RT), which is not present in crystal structures of RT without an inhibitor. Recent studies showed that monomeric forms of the p66 and p51 subunits bind efavirenz with micromolar affinity. The effect of efavirenz on the solution conformations of p66 and p51 monomers was studied by hydrogen-deuterium exchange mass spectrometry (HXMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS)...
December 14, 2010: Biochemistry
Steven M Damo, Aaron H Phillips, Anisa L Young, Sheng Li, Virgil L Woods, David E Wemmer
A fragment of the prion protein, PrP(89-143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR...
October 15, 2010: Journal of Biological Chemistry
Jing Fang, Kasper D Rand, Michelle C Silva, Thomas E Wales, John R Engen, Penny J Beuning
The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD'. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent -1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD'(2)C)...
April 23, 2010: Journal of Molecular Biology
William Burkitt, Paula Domann, Gavin O'Connor
Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs...
April 2010: Protein Science: a Publication of the Protein Society
Thomas E Wales, Keith E Fadgen, Geoff C Gerhardt, John R Engen
The conformational properties of proteins can be probed with hydrogen/deuterium exchange mass spectrometry (HXMS). In order to maintain the deuterium label during LC/MS analyses, chromatographic separation must be done rapidly (usually in under 8-10 min) and at 0 degrees C. Traditional RP-HPLC with approximately 3-mum particles has shown generally poor chromatographic performance under these conditions and thereby has been prohibitive for HXMS analyses of larger proteins and many protein complexes. Ultraperformance liquid chromatography (UPLC) employs particles smaller than 2 mum in diameter to achieve superior resolution, speed, and sensitivity as compared to HPLC...
September 1, 2008: Analytical Chemistry
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