Determination of Histidine pK_a Values in the Propeptides of Furin and Proprotein Convertase 1/3 Using Histidine Hydrogen–Deuterium Exchange Mass Spectrometry

Abstract

Propeptides of proprotein convertases regulate activation of their protease domains by sensing the organellar pH within the secretory pathway. Earlier experimental work highlighted the importance of a conserved histidine residue within the propeptide of a widely studied member, furin. A subsequent evolutionary analysis found an increase in histidine content within propeptides of secreted eukaryotic proteases compared with their prokaryotic orthologs. However, furin activates in the trans-golgi network at a pH of 6.5 while a paralog, proprotein convertase 1/3, activates in secretory vesicles at a pH of 5.5. It is unclear how a conserved histidine can mediate activation at two different pH values. In this manuscript, we measured the pKa values of histidines within the propeptides of furin and proprotein convertase 1/3 using a histidine hydrogen–deuterium exchange mass spectrometry approach. The high density of histidine residues combined with an abundance of basic residues provided challenges for generation of peptide ions with unique histidine residues, which were overcome by employing ETD fragmentation. During this analysis, we found slow hydrogen–deuterium exchange in residues other than histidine at basic pH. Finally, we demonstrate that the pKa of the conserved histidine in proprotein convertase 1/3 is acid-shifted compared with furin and is consistent with its lower pH of activation.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“… 127 Additionally, there are reports that under exhaustive deuterium exchange other carbon–hydrogens can start to exchange, which may further distort the level of deuterium in a harshly treated deuterated sample. 128 , 129 A safer approach is to incubate the protein for a long period of time (12–24 h) at a low pH (between 2.5 and 4) and at room temperature in the presence of denaturants (guanidine or urea). 76 , 130 When preparing a maximally deuterated control, it is important to match the final percentage of deuterium to the other samples in the data set.…”

“…Furthermore, high temperatures can lead to exchange of the C 2 proton on histidine side chains (see section 3.7), which will inflate the levels of observed deuterium uptake observed . Additionally, there are reports that under exhaustive deuterium exchange other carbon–hydrogens can start to exchange, which may further distort the level of deuterium in a harshly treated deuterated sample. , A safer approach is to incubate the protein for a long period of time (12–24 h) at a low pH (between 2.5 and 4) and at room temperature in the presence of denaturants (guanidine or urea). , When preparing a maximally deuterated control, it is important to match the final percentage of deuterium to the other samples in the data set. High urea/guanidine content adds a high concentration of fast-exchanging hydrogens to the solution, which can offset the final percentage of deuterium in the resulting mixture .…”

“…Furthermore, the deuterium label in the C 2 position does not undergo scrambling during CID, making it easy to localize by conventional MS/MS methods. 625 C 2 exchange has since been used for mechanistic studies, elucidating the involvement of specific histidines in proteases, 128,626 and was found to be an effective general approach for assessing protein folding stability and changes in stability upon ligand binding. 627 HDX-MS has been useful for other macromolecules besides proteins and peptides.…”

Nitrogen-doped carbon nanoparticle modulated turn-on fluorescent probes for histidine detection and its imaging in living cells

“…Furthermore, it controls the transmission of metal elements in biological bases and acts as a neurotransmitter or neuromodulator in the central nervous system of mammals. [1][2][3] The abnormal expression level of histidine has been considered as an indicator of many diseases. For example, the low level of histidine in blood plasma may induce rheumatoid arthritis, nerve deafness, liver cirrhosis and pulmonary disease, 4,5 while, the overexpression of histidine is associated with several diseases, such as cancer, AIDS, Alzheimer's disease, and chronic kidney disease.…”

Identifying Protein–Drug Interactions in Cell Lysates Using Histidine Hydrogen Deuterium Exchange

“…His-HDX has been mainly used to determine the p K a and solvent accessibility of histidine residues in proteins ,− but also to measure the thermodynamic stability of protein–metal and protein–protein interactions. , The measurement of the thermodynamic stability of proteins has been carried out by incubating proteins of interest in different concentrations of a chemical denaturant like guanidine-HCl in deuterium oxide solvent and monitoring the extent of deuterium incorporation into His residues that are protected from the solvent in the proteins’ native structure but exposed to the solvent upon denaturation. The His-HDX based method is anticipated to have two significant advantages over the other stability-based methods mentioned above when applied to complex protein samples like cell lysates.…”