Opening opportunities for Kd determination and screening of MHC peptide complexes

Kopicki, Janine-Denise; Saikia, Ankur; Niebling, Stephan; Günther, Christian; Anjanappa, Raghavendra; García-Alai, María; Springer, Sebastian; Uetrecht, Charlotte

doi:10.1038/s42003-022-03366-0

Cited by 7 publications

(7 citation statements)

References 66 publications

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“…Experimental mass, area under the curve (AUC) and visualization was evaluated with Unidec (Michael T. Marty [28]). Kd determination was calculated after [64].…”

Section: Data Analysis For Native Msmentioning

confidence: 99%

An RNA to rule them all: Critical steps in Lassa virus ribonucleoparticle assembly and recruitment

Saenger

Williams

et al. 2023

Preprint

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Lassa virus is a negative-strand RNA virus with only four structural proteins that causes periodic outbreaks in West Africa. The nucleoprotein (NP) encapsidates the viral genome, forming the ribonucleoparticles together with the viral RNA and the L protein, which have to be continuously restructured during viral genome replication and transcription. Z protein is important for ribonucleoparticle recruitment, viral particle assembly and budding, and has also been shown to interact with the L protein. However, the interaction of NP, viral RNA and Z is poorly understood. Here, we characterize the interactions between Lassa virus NP, Z and RNA using structural mass spectrometry. We identify the presence of RNA as the driver for disassembly of ring-like NP trimers into monomers to subsequently form higher order assemblies. We locate the interaction site of Z and NP and demonstrate that while NP binds Z independently of the presence of RNA, this interaction is pH-dependent. These data improve our understanding of ribonucleoparticle assembly and recruitment.

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“…Experimental mass, area under the curve (AUC) and visualization was evaluated with Unidec (Michael T. Marty [28]). Kd determination was calculated after [64].…”

Section: Data Analysis For Native Msmentioning

confidence: 99%

An RNA to rule them all: Critical steps in Lassa virus ribonucleoparticle assembly and recruitment

Saenger

Williams

et al. 2023

Preprint

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“…First, we reasoned that an increased k off , at a rather stable k on , should be accompanied by an increase in the equilibrium dissociation constant ( K D ), since

We, therefore, measured the K D by peptide titration with steady‐state anisotropy 29 and found that the pH did not significantly affect the peptide binding affinity of the complexes (Figure 2c,d ). Second, another readout of peptide binding affinity to MHC‐I is the thermal stability of the peptide‐MHC‐I complexes 20 , 30 , 31 , 32 , 33 , 34 , 35 ; to measure it, we used nanoscale differential scanning fluorimetry (nanoDSF), which quantifies protein unfolding by the change in the fluorescence emission of tryptophan indoles and generates a thermal denaturation curve, the midpoint of which corresponds to the melting temperature ( T m ). 17 We found that there was little influence of the pH on the T m of high‐affinity peptide complexes with wtA2 or wtA24, whereas for medium‐affinity peptides, there was a slight stabilization at low pH (Figure 2e ).…”

Section: Resultsmentioning

confidence: 99%

Fast peptide exchange on major histocompatibility complex class I molecules by acidic stabilization of a peptide‐empty intermediate

et al. 2022

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The cell biology and biochemistry of peptide exchange on major histocompatibility complex class I (MHC‐I) proteins are of great interest in the study of immunodominance, which requires iterative optimization of peptide affinity, and cross‐presentation of pathogen and tumor antigens, in which endogenous peptides are exchanged for exogenous ones. Even though several methods exist to catalyze peptide exchange on recombinant MHC‐I proteins, the cellular conditions and mechanisms allowing for peptide exchange in vivo remain unclear. Here, we demonstrate that low pH, as present in endosomes, indeed triggers peptide exchange, and we dissect the individual steps of the exchange reaction. We find that low pH stabilizes the peptide‐empty forms of MHC‐I that occur as intermediates of the exchange reaction, and that is synergizes with dipeptides and with disulfide‐mediated stabilization of MHC‐I.

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“…Additionally, this allows to investigate complexes otherwise not accessible by the used MS method, either due to their ionization efficiency, their mass or due to dissociation during ionization (Wortmann et al, 2008;El-Hawiet et al, 2010). Kopicki et al were recently able to even make use of a contaminant binding to the Major Histocompatibility Complex (MHC) that is displaced by binding peptides for affinity screening (Kopicki et al, 2022). The use of a well-defined reference system can furthermore be utilized for affinity screening, as shown by Kitov et al with their competitive universal proxy receptor assay (CUPRA) (Kitov et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Determination of dissociation constants via quantitative mass spectrometry

Schulte

Tants²,

Ehr³

et al. 2023

Front. Anal. Sci.

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The interplay of biomolecules governs all cellular processes. Qualitative analysis of such interactions between biomolecules as well as the quantitative assessment of their binding affinities are essential for the understanding of biochemical mechanisms. As scientific interest therefore moves beyond pure structural investigation, methods that allow for the investigation of such interactions become increasingly relevant. In this perspective we outline classical methods that are applicable for the determination of binding constants and highlight specifically mass spectrometry based methods. The use of mass spectrometry to gain quantitative information about binding affinities however is a still developing field. Here, we discuss different approaches, which emerged over the last years to determine dissociation constants (KD) with mass spectrometry based methods. Specifically, we highlight the recent development of quantitative Laser Induced Liquid Bead Ion Desorption (qLILBID) mass spectrometry for the example of double stranded deoxyribonucleic acids as well as for different RNA—RNA binding protein systems. We show that quantitative laser induced liquid bead ion desorption can successfully be used for the top down investigation of complexes and their dissociation constants values ranging from low nM to low µM affinities.

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Opening opportunities for Kd determination and screening of MHC peptide complexes

Cited by 7 publications

References 66 publications

An RNA to rule them all: Critical steps in Lassa virus ribonucleoparticle assembly and recruitment

An RNA to rule them all: Critical steps in Lassa virus ribonucleoparticle assembly and recruitment

Fast peptide exchange on major histocompatibility complex class I molecules by acidic stabilization of a peptide‐empty intermediate

Determination of dissociation constants via quantitative mass spectrometry

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