Biology subjects

Kipping, M.

Publications and source records attributed to Kipping, M..

4 recordsLinked to original sources

bioRxiv2022

Structural Assessment of the Full-Length Wild-Type Tumor Suppressor Protein p53 by Mass Spectrometry-Guided Computational Modeling

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53s C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with AI-based computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53s C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

biochemistry↗

bioRxiv2022

First direct observation of 'elongated' conformational states in α-synuclein upon liquid-liquid phase separation

-Synuclein (-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewys bodies in neurons, which are the hallmark of Parkinsons Disease (PD). Neurotoxicity precedes the formation of aggregates and is probably related to LLPS of -syn in the cell. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into -syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). COMPASS unravels transient interactions between -syn molecules in liquid droplets. In this work, we show that the conformational ensemble of -syn shifts from a hairpin-like structure towards more elongated conformational states upon LLPS. We obtain insights into the critical initial stages of PD and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

biochemistry↗

bioRxiv2021

Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 508 unique cross-linking sites for BSA, 868 for the E. coli ribosome, and 1,623 unique cross-links for nuclear lysates, corresponding to 1,088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/437136v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@141c418org.highwire.dtl.DTLVardef@b13483org.highwire.dtl.DTLVardef@1eeb0ecorg.highwire.dtl.DTLVardef@1a15c12_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

First 3D-Structural Data of Full-length Rod-Outer-Segment Guanylyl Cyclase 1 in Bovine Retina by Cross-linking/Mass Spectrometry

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 in bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data reveal that the -helical domain connecting the kinase homology and catalytic domains can acquire different conformations. Also, the XL-MS data of native ROS-GC1 in bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

biochemistry↗