Publications by authors named "Kasper D Rand"

75 Publications

Probing the Conformational Dynamics of Affinity-Enhanced T Cell Receptor Variants upon Binding the Peptide-Bound Major Histocompatibility Complex by Hydrogen/Deuterium Exchange Mass Spectrometry.

Biochemistry 2021 Mar 9;60(11):859-872. Epub 2021 Mar 9.

Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark.

Binding of the T cell receptor (TCR) to its cognate, peptide antigen-loaded major histocompatibility complex (pMHC) is a key interaction for triggering T cell activation and ultimately elimination of the target cell. Despite the importance of this interaction for cellular immunity, a comprehensive molecular understanding of TCR specificity and affinity is lacking. We conducted hydrogen/deuterium exchange mass spectrometry (HDX-MS) analyses of individual affinity-enhanced TCR variants and clinically relevant pMHC class I molecules (HLA-A*0201/NY-ESO-1) to investigate the causality between increased binding affinity and conformational dynamics in TCR-pMHC complexes. Differential HDX-MS analyses of TCR variants revealed that mutations for affinity enhancement in TCR CDRs altered the conformational response of TCR to pMHC ligation. Improved pMHC binding affinity was in general observed to correlate with greater differences in HDX upon pMHC binding in modified TCR CDR loops, thereby providing new insights into the TCR-pMHC interaction. Furthermore, a specific point mutation in the β-CDR3 loop of the NY-ESO-1 TCR associated with a substantial increase in binding affinity resulted in a substantial change in pMHC binding kinetics (i.e., very slow , revealed by the detection of EX1 HDX kinetics), thus providing experimental evidence for a slow induced-fit binding mode. We also examined the conformational impact of pMHC binding on an unrelated TRAV12-2 gene-encoded TCR directed against the immunodominant MART-1 cancer antigen restricted by HLA-A*0201. Our findings provide a molecular basis for the observed TRAV12-2 gene bias in natural CD8 T cell-based immune responses against the MART-1 antigen, with potential implications for general ligand discrimination and TCR cross-reactivity processes.
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http://dx.doi.org/10.1021/acs.biochem.1c00035DOI Listing
March 2021

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

J Am Soc Mass Spectrom 2021 Mar 8. Epub 2021 Mar 8.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen O, Denmark.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.
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http://dx.doi.org/10.1021/jasms.0c00431DOI Listing
March 2021

Thiol-ene microfluidic chip for fast on-chip sample clean-up, separation and ESI mass spectrometry of peptides and proteins.

Anal Chim Acta 2020 Dec 12;1140:168-177. Epub 2020 Oct 12.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100, Copenhagen O, Denmark. Electronic address:

Mass spectrometry (MS) is a key technology for sensitive and high-resolution mass analysis of peptides and proteins. Sample clean-up and chromatographic separation is typically performed prior to MS analysis to limit adduct formation and ionization suppression. Usually, this requires a high-pressure LC pump system equipped with expensive metal chromatographic columns placed in-line of an electrospray ionization (ESI) source. Microfluidic devices coupled to MS have gained considerable attention, due to the promise of low manufacturing costs, low sample consumption and channels with a high surface area to volume ratio and tailorable functional groups. Here, we describe a thiol-ene microfluidic chip capable of fast chromatographic sample clean-up, concentration, and separation of complex protein and peptide mixtures with direct on-chip ESI. On-chip reversed-phase chromatography (RPC) was performed through an in-situ polymerized monolith frit for retaining inexpensive commercially available reversed-phase (RP) spherical particles, while on-chip ESI is achieved through an emitter monolithically implemented by precision micro milling. The on-chip integration of both RPC and ESI emitter allowed for a minimization of dead-volumes and enables very fast sample clean-up, efficient ionization, and mass analysis of peptides and proteins from complex matrices.
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http://dx.doi.org/10.1016/j.aca.2020.09.062DOI Listing
December 2020

Deglycosylation by the Acidic Glycosidase PNGase H Enables Analysis of N-Linked Glycoproteins by Hydrogen/Deuterium Exchange Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Nov 5;31(11):2305-2312. Epub 2020 Oct 5.

Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become an important method to study the structural dynamics of proteins. However, glycoproteins represent a challenge to the traditional HDX-MS workflow for determining the deuterium uptake of the protein segments that contain the glycan. We have recently demonstrated the utility of the glycosidase PNGase A to enable HDX-MS analysis of N-glycosylated protein regions. Here, we have investigated the use of the acidic glycosidase PNGase H, which has a pH optimum at 2.6, to efficiently deglycosylate N-linked glycosylated peptides during HDX-MS analysis of glycoproteins. Our results show that PNGase H retains high deglycosylation activity at HDX quench conditions. When used in an HDX-MS workflow, PNGase H allowed the extraction of HDX data from all five glycosylated regions of the serpin α-antichymotrypsin. We demonstrate that PNGase A and PNGase H are capable of similar deglycosylation performance during HDX-MS analysis of α-antichymotrypsin and the IgG1 antibody trastuzumab (TZ). However, PNGase H provides broader specificity and greater tolerance to the disulfide-bond reducing agent TCEP, while PNGase A offers advantages in terms of commercial availability and purity. Overall, our findings demonstrate the unique features of PNGase H for improving conformational analysis of glycoproteins by HDX-MS, in particular, challenging glycoproteins containing both glycosylations and disulfide bonds.
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http://dx.doi.org/10.1021/jasms.0c00258DOI Listing
November 2020

Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis.

PeerJ 2020 26;8:e9408. Epub 2020 Jun 26.

Structural Biology and NMR Laboratory, Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen N, Denmark.

Background: Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown.

Methods: Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals.

Results: The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.
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http://dx.doi.org/10.7717/peerj.9408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323712PMC
June 2020

Probing the conformational impact of detergents on the integral membrane protein LeuT by global HDX-MS.

J Proteomics 2020 08 30;225:103845. Epub 2020 May 30.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen O, Denmark. Electronic address:

Neurotransmitter:sodium symporters (NSS) are integral membrane proteins (IMP), responsible for reuptake of neurotransmitters from the synaptic cleft. Due to challenges in production of mammalian NSS in their active form, the prokaryotic hydrophobic amino acid transporter, LeuT, served here as a steadfast model for elucidation of structure-function relationship. As NSS proteins reside within phospholipid bilayer, they require stabilization by artificial membrane systems upon their extraction. Right choice of artificial membrane system is crucial as suboptimal detergent and/or lipids can lead to destabilization or non-native stabilization. Here we study the effect of related detergents, dodecyl maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG), on the conformational dynamics of LeuT by global HDX-MS, in the presence of functionally relevant ligands. We observed that LeuT is more dynamic when solubilized in DDM compared to LMNG. Moreover, LeuT exhibited increased HDX in the presence of K compared to Na, indicating a more dynamic conformation in the presence of K. Upon addition of leucine, LeuT underwent additional stabilization relative to the Na-bound state. Finally, peak broadening was observed, suggesting that LeuT undergoes slow unfolding/refolding dynamics in detergent solution. These slow dynamics were verified by local HDX, also proving that detergents modulate the rate of these dynamics. SIGNIFICANCE: Overall, we show the efficacy of global HDX-MS to evaluate the effect of artificial membrane systems on integral membrane proteins and the importance of carefully selecting compatible detergent (and/or lipid) for the solubilization of this class of proteins.
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http://dx.doi.org/10.1016/j.jprot.2020.103845DOI Listing
August 2020

Hydrogen/deuterium exchange mass spectrometry with improved electrochemical reduction enables comprehensive epitope mapping of a therapeutic antibody to the cysteine-knot containing vascular endothelial growth factor.

Anal Chim Acta 2020 Jun 10;1115:41-51. Epub 2020 Apr 10.

Department of Pharmacy, Copenhagen University, Universitetsparken 2, Denmark. Electronic address:

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become a popular method for analysis of the conformational dynamics and interactions of proteins. Disulfide-bonded proteins, however, present a challenge to HDX-MS as they require efficient disulfide bond reduction prior to enzymatic proteolysis. Electrochemical reduction (ER) provides an attractive solution to tackle disulfide-bonded proteins that are resistant to conventional chemical reduction during HDX-MS. However, ER-enabled HDX-MS has been limited by technical challenges including partial unwanted protein oxidation side-reactions, incompatibility with certain buffer components and most importantly, a lack of overall method robustness. In this study, we have sought to address these challenges. We perform a systematic screening of the compatibility of ER to buffers commonly used in HDX-MS samples by using a reliable and simple system suitability test (SST). Furthermore, we demonstrate the benefits of a new design of the electrochemical cell (EC) for ER-enabled HDX-MS, which include a) high repeatability and robustness over large sample batches without the need for electrode polishing and b) high reduction efficiency of disulfide-bonded proteins without unwanted oxidation side-reactions. We show the real-world applicability of the optimized ER-enabled HDX-MS workflow by performing an epitope mapping of a Fab fragment of a therapeutic monoclonal antibody (mAb) to the cysteine knot-containing vascular endothelial growth factor (VEGF). The results allow us to comprehensively map sites in VEGF involved in mAb binding. Overall, our findings show how ER and HDX-MS can be combined to enable analysis of the conformation and interactions of challenging disulfide-rich proteins.
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http://dx.doi.org/10.1016/j.aca.2020.04.014DOI Listing
June 2020

Investigating surrogate cerebrospinal fluid matrix compositions for use in quantitative LC-MS analysis of therapeutic antibodies in the cerebrospinal fluid.

Anal Bioanal Chem 2020 Mar 1;412(7):1653-1661. Epub 2020 Feb 1.

Translational DMPK, H. Lundbeck A/S, Ottiliavej 9, 2500, Valby, Denmark.

As quantitative analysis of biotherapeutics in cerebrospinal fluid (CSF) with LC-MS becomes increasingly widespread, there is a need for method developments towards higher sensitivity. By using artificial CSF (aCSF) in the development phase, the consumption of costly and sparsely available CSF can be limited. The aCSF compositions tested here were made from various dilutions of bovine serum albumin (BSA) or rat plasma to mimic the total protein concentration found in CSF. Focusing on monoclonal antibodies, the aCSF was spiked with human immunoglobulin (hIgG) and prepared with the bottom-up analysis technique using LC-MS. Assuming that the composition of the aCSF would affect the digest, the response from aCSF matrices was compared with CSF from rat, monkey, and dog in terms of estimated sample concentration and matrix effects. The samples were spiked with hIgG in the range of 10 to 1000 ng/mL and volumes of 10 μL were transferred to sample preparation. The results indicate that BSA dilutions from 300 to 2000 μg/mL and rat plasma dilutions of 0.5-2% provide the most accurate concentration estimates when compared with rat CSF. 1000 μg/mL BSA did not produce significantly different concentration estimates for 500 ng/mL samples when compared with CSF from rat, monkey, and dog, and can therefore be used as aCSF for several different species.
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http://dx.doi.org/10.1007/s00216-020-02403-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026242PMC
March 2020

Benzisothiazolinone Derivatives as Potent Allosteric Monoacylglycerol Lipase Inhibitors That Functionally Mimic Sulfenylation of Regulatory Cysteines.

J Med Chem 2020 02 2;63(3):1261-1280. Epub 2019 Dec 2.

Dipartimento di Scienze degli Alimenti e del Farmaco , Università degli Studi di Parma , Parco Area delle Scienze 27/A , I-43124 Parma , Italy.

We describe a set of benzisothiazolinone (BTZ) derivatives that are potent inhibitors of monoacylglycerol lipase (MGL), the primary degrading enzyme for the endocannabinoid 2-arachidonoyl--glycerol (2-AG). Structure-activity relationship studies evaluated various substitutions on the nitrogen atom and the benzene ring of the BTZ nucleus. Optimized derivatives with nanomolar potency allowed us to investigate the mechanism of MGL inhibition. Site-directed mutagenesis and mass spectrometry experiments showed that BTZs interact in a covalent reversible manner with regulatory cysteines, Cys201 and Cys208, causing a reversible sulfenylation known to modulate MGL activity. Metadynamics simulations revealed that BTZ adducts favor a closed conformation of MGL that occludes substrate recruitment. The BTZ derivative protected neuronal cells from oxidative stimuli and increased 2-AG levels in the mouse brain. The results identify Cys201 and Cys208 as key regulators of MGL function and point to the BTZ scaffold as a useful starting point for the discovery of allosteric MGL inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01679DOI Listing
February 2020

Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

Anal Chem 2019 09 13;91(17):10970-10978. Epub 2019 Aug 13.

Department of Pharmacy , University of Copenhagen , Universitetsparken 2 , Copenhagen E DK-2100 , Denmark.

Insight into the structure-function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl/H exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MS-compatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence; however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.
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http://dx.doi.org/10.1021/acs.analchem.9b00973DOI Listing
September 2019

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Nat Methods 2019 07 27;16(7):595-602. Epub 2019 Jun 27.

Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
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http://dx.doi.org/10.1038/s41592-019-0459-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614034PMC
July 2019

Substrate-induced conformational dynamics of the dopamine transporter.

Nat Commun 2019 06 20;10(1):2714. Epub 2019 Jun 20.

Laboratory for Membrane Protein Dynamics, Department of Neuroscience, University of Copenhagen, 2200, Copenhagen N, Denmark.

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
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http://dx.doi.org/10.1038/s41467-019-10449-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586795PMC
June 2019

Investigating the Conformational Response of the Sortilin Receptor upon Binding Endogenous Peptide- and Protein Ligands by HDX-MS.

Structure 2019 07 16;27(7):1103-1113.e3. Epub 2019 May 16.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen, Denmark. Electronic address:

Sortilin is a multifunctional neuronal receptor involved in sorting of neurotrophic factors and apoptosis signaling. So far, structural characterization of sortilin and its endogenous ligands has been limited to crystallographic studies of sortilin in complex with the neuropeptide neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of sortilin to binding biological ligands including the peptides neurotensin and the sortilin propeptide and the proteins progranulin and pro-nerve growth factor-β. The results show that the ligands use two binding sites inside the cavity of the β-propeller of sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of sortilin. Our findings improve our structural understanding of sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.
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http://dx.doi.org/10.1016/j.str.2019.04.006DOI Listing
July 2019

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

Anal Chem 2019 06 22;91(11):6953-6961. Epub 2019 May 22.

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram , The Hebrew University of Jerusalem , Jerusalem 91904 , Israel.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
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http://dx.doi.org/10.1021/acs.analchem.9b00658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625963PMC
June 2019

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Anal Chem 2019 06 14;91(11):7336-7345. Epub 2019 May 14.

Department of Chemistry , University of Kansas , 1567 Irving Hill Road , Lawrence , Kansas 66045 , United States.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ s⟩ ≤ (0.15 ± 0.01) Da (1σ). All laboratories achieved ⟨s⟩ ≤ 0.4 Da. For immersions of protein at T = (3.6 to 25) °C and for DO exchange times of t = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σ( t) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at T = 25 °C exhibited reproducibility of σ( t) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.
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http://dx.doi.org/10.1021/acs.analchem.9b01100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745711PMC
June 2019

Conformational dynamics of the human serotonin transporter during substrate and drug binding.

Nat Commun 2019 04 11;10(1):1687. Epub 2019 Apr 11.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Copenhagen, 2100, Denmark.

The serotonin transporter (SERT), a member of the neurotransmitter:sodium symporter family, is responsible for termination of serotonergic signaling by re-uptake of serotonin (5-HT) into the presynaptic neuron. Its key role in synaptic transmission makes it a major drug target, e.g. for the treatment of depression, anxiety and post-traumatic stress. Here, we apply hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs. Our results reveal significant changes in dynamics in regions TM1, EL3, EL4, and TM12 upon binding co-transported ions (Na/K) and ligand-mediated changes in TM1, EL3 and EL4 upon binding 5-HT, the drugs S-citalopram, cocaine and ibogaine. Our results provide a comprehensive direct view of the conformational response of SERT upon binding both biologically relevant substrate/ions and ligands of pharmaceutical interest, thus advancing our understanding of the structure-function relationship in SERT.
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http://dx.doi.org/10.1038/s41467-019-09675-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459873PMC
April 2019

A post-translational modification of human Norovirus capsid protein attenuates glycan binding.

Nat Commun 2019 03 21;10(1):1320. Epub 2019 Mar 21.

Institute of Chemistry and Metabolomics, Center of Structural and Cell Biology in Medicine (CSCM), University of Lübeck, Ratzeburger Allee 160, 23562, Lübeck, Germany.

Attachment of human noroviruses to histo blood group antigens (HBGAs) is essential for infection, but how this binding event promotes the infection of host cells is unknown. Here, we employ protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P-domain of a prevalent virus strain (GII.4). We report a highly selective transformation of asparagine 373, located in an antigenic loop adjoining the HBGA binding site, into an iso-aspartate residue. This spontaneous post-translational modification (PTM) proceeds with an estimated half-life of a few days at physiological temperatures, independent of the presence of HBGAs but dramatically affecting HBGA recognition. Sequence conservation and the surface-exposed position of this PTM suggest an important role in infection and immune recognition for many norovirus strains.
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http://dx.doi.org/10.1038/s41467-019-09251-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428809PMC
March 2019

Molecular architecture of the Jumonji C family histone demethylase KDM5B.

Sci Rep 2019 03 11;9(1):4019. Epub 2019 Mar 11.

Biostructural Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 162, 2100, Copenhagen, Denmark.

The full length human histone 3 lysine 4 demethylase KDM5B (PLU-1/Jarid1B) has been studied using Hydrogen/Deuterium exchange mass spectrometry, homology modelling, sequence analysis, small angle X-ray scattering and electron microscopy. This first structure on an intact multi-domain Jumonji histone demethylase reveal that the so-called PLU region, in the central region of KDM5B, has a curved α-helical three-dimensional structure, that acts as a rigid linker between the catalytic core and a region comprising four α-helices, a loop comprising the PHD2 domain, two large intrinsically disordered loops and the PHD3 domain in close proximity. The dumbbell shaped and curved KDM5B architecture observed by electron microscopy is complementary to the nucleosome surface and has a striking overall similarity to that of the functionally related KDM1A/CoREST complex. This could suggest that there are similarities between the demethylation mechanisms employed by the two histone 3 lysine 4 demethylases at the molecular level.
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http://dx.doi.org/10.1038/s41598-019-40573-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411775PMC
March 2019

Thiol-ene Microfluidic Chip for Performing Hydrogen/Deuterium Exchange of Proteins at Subsecond Time Scales.

Anal Chem 2019 01 21;91(2):1309-1317. Epub 2018 Dec 21.

Protein Analysis Group, Department of Pharmacy , University of Copenhagen , 2100 Copenhagen , Denmark.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (subsecond) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photochemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer, and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14-1.1 s time frame. The HDX of Hb at 0.14-1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX and loops undergoing pronounced HDX even at subsecond time scales. On-chip HDX of Hb at time points ranging from 0.14-1.1 s demonstrates the ability to distinguish fast exchanging amides and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution.
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http://dx.doi.org/10.1021/acs.analchem.8b03050DOI Listing
January 2019

Structure and Dynamics of a Promiscuous Xanthan Lyase from Paenibacillus nanensis and the Design of Variants with Increased Stability and Activity.

Cell Chem Biol 2019 02 29;26(2):191-202.e6. Epub 2018 Nov 29.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Copenhagen 2100, Denmark. Electronic address:

We have characterized the structure and dynamics of the carbohydrate-modifying enzyme Paenibacillus nanensis xanthan lyase (PXL) involved in the degradation of xanthan by X-ray crystallography, small-angle X-ray scattering, and hydrogen/deuterium exchange mass spectrometry. Unlike other xanthan lyases, PXL is specific for both unmodified mannose and pyruvylated mannose, which we find is correlated with structural differences in the substrate binding groove. The structure of the full-length enzyme reveals two additional C-terminal modules, one of which belongs to a new non-catalytic carbohydrate binding module family. Ca are critical for the activity and conformation of PXL, and we show that their removal by chelating agents results in localized destabilization/unfolding of particularly the C-terminal modules. We use the structure and the revealed impact of Ca coordination on conformational dynamics to guide the engineering of PXL variants with increased activity and stability in a chelating environment, thus expanding the possibilities for industrial applications of PXL.
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http://dx.doi.org/10.1016/j.chembiol.2018.10.016DOI Listing
February 2019

Probing the Dissociation of Protein Complexes by Means of Gas-Phase H/D Exchange Mass Spectrometry.

J Am Soc Mass Spectrom 2019 Jan 20;30(1):45-57. Epub 2018 Nov 20.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-2064-1DOI Listing
January 2019

Side-chain moieties from the N-terminal region of Aβ are Involved in an oligomer-stabilizing network of interactions.

PLoS One 2018 6;13(8):e0201761. Epub 2018 Aug 6.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

Oligomeric forms of the Aβ peptide represent the most probable neurotoxic agent in Alzheimer's disease. The dynamic and heterogeneous character of these oligomers makes their structural characterization by classic methods difficult. Native mass spectrometry, when supported by additional gas phase techniques, like ion mobility separation and hydrogen-deuterium exchange (IM-HDX-MS), enable analysis of different oligomers coexisting in the sample and may provide species-specific structural information for each oligomeric form populated in the gas phase. Here, we have combined these three techniques to obtain insight into the structural properties of oligomers of Aβ1-40 and two variants with scrambled sequences. Gas-phase HDX-MS revealed a sequence-specific engagement of the side-chains of residues located at the N-terminal part of the peptide in a network of oligomer-stabilizing interactions. Oligomer-specific interactions were no longer observed in the case of the fully scrambled sequence. Also, the ability to form alternative structures, observed for WT Aβ peptide, was lost upon scrambling. Our data underscore a role for the N-terminal residues in shaping the equilibria of oligomeric forms. Although the peptide lacking the N-terminal 1-16 residues (p3 peptide) is thought to be benign, the role of the N-terminus has not been sufficiently characterized yet. We speculate that the interaction networks revealed here may be crucial for enabling structural transitions necessary to obtain mature parallel cross-β structures from smaller antiparallel oligomers. We provide a hypothetical molecular model of the trajectory that allows a gradual conversion from antiparallel to parallel oligomers without decomposition of oligomers. Oligomer-defining interactions involving the Aβ peptide N-terminus may be important in production of the neurotoxic forms and thus should not be neglected.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201761PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6078298PMC
January 2019

Investigating the utility of minimized sample preparation and high-resolution mass spectrometry for quantification of monoclonal antibody drugs.

J Pharm Biomed Anal 2018 Sep 30;159:384-392. Epub 2018 Jul 30.

Department of Pharmacy, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark. Electronic address:

Determination of the pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) is essential for their successful development as drugs. For this purpose, besides the traditional ligand binding assay (LBA), LC-MS/MS method using low resolution mass spectrometers (e.g. triple quadrupole (QqQ)) has become routinely used, however, complicated and lengthy sample pre-treatment (employing immuno-affinity) is often necessary for obtaining sufficient sensitivity and selectivity. In this study, we investigate the capabilities of high-resolution MS instruments for circumventing the complex sample preparation currently needed for sensitive LC-MS/MS-based quantification of mAbs. Employing a simple one-step sample pre-treatment workflow, we compare the ability of three different LC-MS platforms for absolute quantification of a representative monoclonal antibody Rendomab-B1 in serum and plasma. The samples are subjected to protein precipitation with methanol, followed by pellet digestion with trypsin prior to LC-MS analysis. AQUA peptides based on two surrogate mAb peptides selected from an extensive in-silico and experimental screening are used as internal standards. MS/MS acquisitions are developed and systematically examined for 1) a low-resolution QqQ operated in selected reaction monitoring (SRM) acquisition mode, 2) a high-resolution hybrid Quadrupole-Orbitrap (Q-Orbitrap) operated in parallel reaction monitoring (PRM) acquisition mode and 3) a high-resolution hybrid Quadrupole-Time-of-flight (Q-TOF) operated in SRM acquisition mode with enhanced duty cycle (EDC) function. The sensitivity of the high-resolution Q-Orbitrap and Q-TOF methods was significantly higher (LOD of 80 ng/mL) in serum/plasma samples than the low-resolution QqQ method. Finally, the real-world utility of the developed high-resolution MS method with minimized sample handling was demonstrated and validated by determining the PK profile of Rendomab-B1 in mice by a 10-point in vivo study over 15 days.
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http://dx.doi.org/10.1016/j.jpba.2018.07.012DOI Listing
September 2018

Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT.

Sci Adv 2018 05 11;4(5):eaar6179. Epub 2018 May 11.

Protein Analysis Group, Department of Pharmacy, University of Copenhagen, 2100 Copenhagen O, Denmark.

LeuT, a prokaryotic member of the neurotransmitter:sodium symporter (NSS) family, is an established structural model for mammalian NSS counterparts. We investigate the substrate translocation mechanism of LeuT by measuring the solution-phase structural dynamics of the transporter in distinct functional states by hydrogen/deuterium exchange mass spectrometry (HDX-MS). Our HDX-MS data pinpoint LeuT segments involved in substrate transport and reveal for the first time a comprehensive and detailed view of the dynamics associated with transition of the transporter between outward- and inward-facing configurations in a Na- and K-dependent manner. The results suggest that partial unwinding of transmembrane helices 1/5/6/7 drives LeuT from a substrate-bound, outward-facing occluded conformation toward an inward-facing open state. These hitherto unknown, large-scale conformational changes in functionally important transmembrane segments, observed for LeuT in detergent-solubilized form and when embedded in a native-like phospholipid bilayer, could be of physiological relevance for the translocation process.
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http://dx.doi.org/10.1126/sciadv.aar6179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947982PMC
May 2018

Installation, validation, and application examples of two instrumental setups for gas-phase HDX-MS analysis of peptides and proteins.

Methods 2018 07 18;144:113-124. Epub 2018 May 18.

Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry in a millisecond timeframe after ESI (gas-phase HDX-MS) is a fast and sensitive, yet unharnessed method to analyze the primary- and higher-order structure, intramolecular and intermolecular interactions, surface properties, and charge location of peptides and proteins. During a gas-phase HDX-MS experiment, heteroatom-bound non-amide hydrogens are made to exchange with deuterium during a millisecond timespan after electrospray ionization (ESI) by reaction with the highly basic reagent ND, enabling conformational analysis of protein states that are pertinent to the native solution-phase. Here, we describe two different instrumental approaches to enable gas-phase HDX-MS for analysis of peptides and proteins on high-resolution Q-TOF mass spectrometers. We include a description of the procedure and equipment required for successful installation as well as suggested procedures for testing, validation, and troubleshooting of a gas-phase HDX-MS setup. In the two described approaches, gas-phase HDX-MS are performed either immediately after ESI in the cone exit region by leading N-gas over a deuterated ND/DO solution, or by leading purified ND-gas into different traveling wave ion guides (TWIG) of the mass spectrometer. We envision that a detailed description of the two gas-phase HDX-MS setups and their practical implementation and validation can pave the way for gas-phase HDX-MS to become a more routinely used MS technique for structural analysis of peptides and proteins.
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http://dx.doi.org/10.1016/j.ymeth.2018.05.002DOI Listing
July 2018

Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix.

Biochemistry 2018 02 9;57(8):1326-1337. Epub 2018 Feb 9.

Center for Integrated Protein Science Munich (CIPSM) at the Lehrstuhl Chemie der Biopolymere, Technical University of Munich , Weihenstephaner Berg 3, 85354 Freising, Germany.

Flexible transmembrane helices frequently support the conformational transitions between different functional states of membrane proteins. While proline is well known to distort and destabilize transmembrane helices, the role of glycine is still debated. Here, we systematically investigated the effect of glycine on transmembrane helix flexibility by placing it at different sites within the otherwise uniform leucine/valine repeat sequence of the LV16 model helix. We show that amide deuterium/hydrogen exchange kinetics are increased near glycine. Molecular dynamics simulations reproduce the measured exchange kinetics and reveal, at atomic resolution, a severe packing defect at glycine that enhances local hydration. Furthermore, glycine alters H-bond occupancies and triggers a redistribution of α-helical and 3-helical H-bonds. These effects facilitate local helix bending at the glycine site and change the collective dynamics of the helix.
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http://dx.doi.org/10.1021/acs.biochem.7b01197DOI Listing
February 2018

UV Photodissociation Mass Spectrometry Accurately Localize Sites of Backbone Deuteration in Peptides.

Anal Chem 2018 01 28;90(2):1077-1080. Epub 2017 Dec 28.

Department of Pharmacy, University of Copenhagen , Universitetsparken 2, 2100 Copenhagen, Denmark.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is now a routinely used technique to inform on protein structure, dynamics, and interactions. Localizing the incorporated deuterium content on a single residue basis increases the spatial resolution of this technique enabling detailed structural analysis. Here, we investigate the use of ultraviolet photodissociation (UVPD) at 213 nm to measure deuterium levels at single residue resolution in HDX-MS experiments. Using a selectively labeled peptide, we show that UVPD occurs without H/D scrambling as the peptide probe accurately retains its solution-phase deuterium labeling pattern. Our results indicate that UVPD provides an attractive alternative to electron mediated dissociation for increasing the spatial resolution of the HDX-MS experiment, capable of yielding high fragmentation efficiency, high fragment ion diversity, and low precursor ion charge-state dependency.
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http://dx.doi.org/10.1021/acs.analchem.7b04683DOI Listing
January 2018

A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene.

Mol Cell 2017 Dec;68(5):860-871.e7

Biomedical Center Munich, Faculty of Medicine, Ludwig-Maximilians-Universität München, Großhaderner Street 9, 82152 Planegg-Martinsried, Germany; Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Butenandt Street 13, 81377 Munich, Germany; Munich Cluster for Systems Neurology, Ludwig-Maximilians-Universität München, Feodor Lynen Street 17, 81377 Munich, Germany. Electronic address:

DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.
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http://dx.doi.org/10.1016/j.molcel.2017.11.019DOI Listing
December 2017

Conformational characterization of nerve growth factor-β reveals that its regulatory pro-part domain stabilizes three loop regions in its mature part.

J Biol Chem 2017 10 10;292(40):16665-16676. Epub 2017 Aug 10.

From the Department of Pharmacy, University of Copenhagen, 2100 Copenhagen E, Denmark and

Nerve growth factor-β (NGF) is essential for the correct development of the nervous system. NGF exists in both a mature form and a pro-form (proNGF). The two forms have opposing effects on neurons: NGF induces proliferation, whereas proNGF induces apoptosis via binding to a receptor complex of the common neurotrophin receptor (p75NTR) and sortilin. The overexpression of both proNGF and sortilin has been associated with several neurodegenerative diseases. Insights into the conformational differences between proNGF and NGF are central to a better understanding of the opposing mechanisms of action of NGF and proNGF on neurons. However, whereas the structure of NGF has been determined by X-ray crystallography, the structural details for proNGF remain elusive. Here, using a sensitive MS-based analytical method to measure the hydrogen/deuterium exchange of proteins in solution, we analyzed the conformational properties of proNGF and NGF. We detected the presence of a localized higher-order structure motif in the pro-part of proNGF. Furthermore, by comparing the hydrogen/deuterium exchange in the mature part of NGF and proNGF, we found that the presence of the pro-part in proNGF causes a structural stabilization of three loop regions in the mature part, possibly through a direct molecular interaction. Moreover, using tandem MS analyses, we identified two -linked and two -linked glycosylations in the pro-part of proNGF. These results advance our knowledge of the conformational properties of proNGF and NGF and help provide a rationale for the diverse biological effects of NGF and proNGF at the molecular level.
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http://dx.doi.org/10.1074/jbc.M117.803320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633128PMC
October 2017

The extraordinary thermal stability of EstA from S. islandicus is independent of post translational modifications.

Protein Sci 2017 Sep 13;26(9):1819-1827. Epub 2017 Jul 13.

Structural Biology and NMR Laboratory and The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, København, Denmark.

Enzymes from thermophilic and hyper-thermophilic organisms have an intrinsic high stability. Understanding the mechanisms behind their high stability will be important knowledge for the engineering of novel enzymes with high stability. Lysine methylation of proteins is prevalent in Sulfolobus, a genus of hyperthermophilic and acidophilic archaea. Both unspecific and temperature dependent lysine methylations are seen, but the significance of this post-translational modification has not been investigated. Here, we test the effect of eliminating in vivo lysine methylation on the stability of an esterase (EstA). The enzyme was purified from the native host S. islandicus as well as expressed as a recombinant protein in E. coli, a mesophilic host that does not code for any machinery for in vivo lysine methylation. We find that lysine mono methylation indeed has a positive effect on the stability of EstA, but the effect is small. The effect of the lysine methylation on protein stability is secondary to that of protein expression in E. coli, as the E. coli recombinant enzyme is compromised both on stability and activity. We conclude that these differences are not attributed to any covalent difference between the protein expressed in hyperthermophilic versus mesophilic hosts.
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http://dx.doi.org/10.1002/pro.3220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563138PMC
September 2017