Biology subjects

Zamel, J.

Publications and source records attributed to Zamel, J..

4 recordsLinked to original sources

bioRxiv2022

The structural basis for the self-inhibition of DNA binding by apo-σ70

The initiation of transcription in Escherichia coli (E. coli) is facilitated by promoter specificity factors, also known as {sigma} factors, which may bind a promoter only as part of a complex with RNA polymerase (RNAP). By performing in vitro cross-linking mass spectrometry (CL-MS) of apo-{sigma}70, we reveal structural features suggesting a compact conformation compared to the known RNAP-bound extended conformation. Then, we validate the existence of the compact conformation using in vivo CL-MS by identifying cross-links similar to those found in vitro, which deviate from the extended conformation only during the stationary phase of bacterial growth. Conclusively, we provide information in support of a compact conformation of apo-{sigma}70 that exists in live cells, which might represent a transcriptionally-inactive form that can be activated upon binding to RNAP.

biophysics↗

bioRxiv2021

Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

Atomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

biochemistry↗

bioRxiv2020

The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

The intrinsically disordered protein, -synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of -synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of -synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Forster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the -synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of -synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

biophysics↗

bioRxiv2019

Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

Formaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein {beta}NAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

biochemistry↗