Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a ...decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.
Non-linear non-renormalization theorems Cao, Weiguang; Herzog, Franz; Melia, Tom ...
The journal of high energy physics,
08/2023, Letnik:
2023, Številka:
8
Journal Article
Recenzirano
Odprti dostop
A
bstract
We study the mixing of operators under renormalization group flow in quantum theories, and prove a non-renormalization theorem at non-linear order. It dictates zeros up to a certain number ...of loops in anomalous dimension tensors that control, for example, the mixing of operators at order dimension six squared into dimension eight. We obtain new results at up to three loops for the mass dimension eight anomalous dimension tensor of
ϕ
4
theory in
D
= 4
−
2
ε
dimensions and verify the zeros predicted by the theorem.
Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of ...peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide‐specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT‐peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than four times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments.
RNA polymerase (Pol) II produces messenger RNA during transcription of protein-coding genes in all eukaryotic cells. The Pol II structure is known at high resolution from X-ray crystallography for ...two yeast species. Structural studies of mammalian Pol II, however, remain limited to low-resolution electron microscopy analysis of human Pol II and its complexes with various proteins. Here we report the 3.4 Å resolution cryo-electron microscopy structure of mammalian Pol II in the form of a transcribing complex comprising DNA template and RNA transcript. We use bovine Pol II, which is identical to the human enzyme except for seven amino-acid residues. The obtained atomic model closely resembles its yeast counterpart, but also reveals unknown features. Binding of nucleic acids to the polymerase involves 'induced fit' of the mobile Pol II clamp and active centre region. DNA downstream of the transcription bubble contacts a conserved 'TPSA motif' in the jaw domain of the Pol II subunit RPB5, an interaction that is apparently already established during transcription initiation. Upstream DNA emanates from the active centre cleft at an angle of approximately 105° with respect to downstream DNA. This position of upstream DNA allows for binding of the general transcription elongation factor DSIF (SPT4-SPT5) that we localize over the active centre cleft in a conserved position on the clamp domain of Pol II. Our results define the structure of mammalian Pol II in its functional state, indicate that previous crystallographic analysis of yeast Pol II is relevant for understanding gene transcription in all eukaryotes, and provide a starting point for a mechanistic analysis of human transcription.
Chemical cross-links identified by mass spectrometry generate distance restraints that reveal low-resolution structural information on proteins and protein complexes. The technology to reliably ...generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations. Here, we introduce three workflows for incorporating distance restraints generated by chemical cross-linking and mass spectrometry into ROSETTA protocols for comparative and de novo modeling and protein-protein docking. We demonstrate that the cross-link validation and visualization software Xwalk facilitates successful cross-link data integration. Besides the protocols we introduce XLdb, a database of chemical cross-links from 14 different publications with 506 intra-protein and 62 inter-protein cross-links, where each cross-link can be mapped on an experimental structure from the Protein Data Bank. Finally, we demonstrate on a protein-protein docking reference data set the impact of virtual cross-links on protein docking calculations and show that an inter-protein cross-link can reduce on average the RMSD of a docking prediction by 5.0 Å. The methods and results presented here provide guidelines for the effective integration of chemical cross-link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology methods.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Kinetochores are multisubunit complexes that assemble on centromeres to bind spindle microtubules and promote faithful chromosome segregation during cell division. A 16-subunit complex named the ...constitutive centromere-associated network (CCAN) creates the centromere-kinetochore interface. CENP-C, a CCAN subunit, is crucial for kinetochore assembly because it links centromeres with the microtubule-binding interface of kinetochores. The role of CENP-C in CCAN organization, on the other hand, had been incompletely understood. In this paper, we combined biochemical reconstitution and cellular investigations to unveil how CENP-C promotes kinetochore targeting of other CCAN subunits. The so-called PEST domain in the N-terminal half of CENP-C interacted directly with the four-subunit CCAN subcomplex CENP-HIKM. We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex. When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.
Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of ...peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.
A
bstract
We renormalize massless scalar effective field theories (EFTs) to higher loop orders and higher orders in the EFT expansion. To facilitate EFT calculations with the R* renormalization ...method, we construct suitable operator bases using Hilbert series and related ideas in commutative algebra and conformal representation theory, including their novel application to off-shell correlation functions. We obtain new results ranging from full one loop at mass dimension twelve to five loops at mass dimension six. We explore the structure of the anomalous dimension matrix with an emphasis on its zeros, and investigate the effects of conformal and orthonormal operators. For the real scalar, the zeros can be explained by a ‘non-renormalization’ rule recently derived by Bern et al. For the complex scalar we find two new selection rules for mixing
n
- and (
n
−
2)-field operators, with
n
the maximal number of fields at a fixed mass dimension. The first appears only when the (
n
−
2)-field operator is conformal primary, and is valid at one loop. The second appears in more generic bases, and is valid at three loops. Finally, we comment on how the Hilbert series we construct may be used to provide a systematic enumeration of a class of evanescent operators that appear at a particular mass dimension in the scalar EFT.
The generation of mathematical models of biological processes, the simulation of these processes under different conditions, and the comparison and integration of multiple data sets are explicit ...goals of systems biology that require the knowledge of the absolute quantity of the system's components. To date, systematic estimates of cellular protein concentrations have been exceptionally scarce. Here, we provide a quantitative description of the proteome of a commonly used human cell line in two functional states, interphase and mitosis. We show that these human cultured cells express at least ∼10 000 proteins and that the quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell. We discuss how protein abundance is linked to function and evolution.
The majority of all proteins expressed in the human osteosarcoma cell line U2OS were absolutely quantified by mass spectrometry. The quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell.
Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central ...role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.
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