Measuring in vivo protein turnover and exchange in yeast macromolecular assemblies.

We present a comprehensive and robust protocol to track the dynamics of all proteins in a complex in yeast cells. A single member of the protein assembly is tagged and conditionally expressed, minimizing the perturbations to the protein complex. Then, SILAC labeling and affinity purification are used for the assessment of the whole protein complex dynamics. This method can determine and distinguish both subunit turnover and exchange specifically in an assembly to provide a comprehensive picture of assembly dynamics. For complete details on the use and execution of this protocol, please refer to Hakhverdyan et al. (2021).

Dissecting the Structural Dynamics of the Nuclear Pore Complex.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.

The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination.

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.

A pipeline for determining protein-protein interactions and proximities in the cellular milieu.

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.

Ferrocenylbutadiyne.

The title compound, [Fe(C(5)H(5))(C(9)H(5))], crystallizes in a form of a π-π-stacked assembly formed as a result of strong inter-molecular π-π inter-actions between (a) the triple bonds of two neighboring butadiyne substituents overlapping in a 'head-to-tail' fashion [characterized by C⋯C short contacts of 3.622 (5), 3.567 (6) and 3.556 (6) Å] and (b) the triple bonds of the butadiyne substituent and substituted cyclo-pendadiene ring of neighboring mol-ecules [C⋯C = 3.474 (5) and 3.492 (6) Å]. The linear butadiyne substituent has alternating C-C triple and single bonds, while the unsubstituted cyclo-penta-diene ring is slightly positionally disordered (although the structure reported here was solved as non-disordered) and retains a close to eclipsed conformation.

Metal-free and transition-metal tetraferrocenylporphyrins part 1: synthesis, characterization, electronic structure, and conformational flexibility of neutral compounds.

H(2)TFcP [TFcP = 5,10,15,20-tetraferrocenyl porphyrin(2-)] was prepared by a direct tetramerization reaction between pyrrole and ferrocene carbaldehyde in the presence of a BF(3) catalyst, while the series of MTFcP (M = Zn, Ni, Co and Cu) were prepared by a metallation reaction between H(2)TFcP and respective metal acetates. All compounds were characterized by UV-vis and MCD spectroscopy, APCI MS and MS/MS methods, high-resolution ESI MS and XPS spectroscopy. Diamagnetic compounds were additionally characterized using (1)H and (13)C NMR methods, while the presence of low-spin iron(ii) centers in the neutral compounds was confirmed by Mössbauer spectroscopy and by analysis of the XPS Fe 2p peaks, revealing equivalent Fe sites. XPS additionally showed the influence on Fe 2p binding energies exerted by the distinct central metal ions. The conformational flexibility of ferrocene substituents in H(2)TFcP and MTFcP, was confirmed using variable-temperature NMR and computational methods. Density functional theory predicts that alpha,beta,alpha,beta atropisomers with ruffled porphyrin cores represent minima on the potential energy surfaces of both H(2)TFcP and MTFcP. The degree of non-planarity is central-metal dependent and follows the trend: ZnTFcP < H(2)TFcP approximately CuTFcP < CoTFcP < NiTFcP. In all cases, a set of occupied, predominantly ferrocene-based molecular orbitals were found between the highest occupied and the lowest unoccupied, predominantly porphyrin-based molecular orbitals. The vertical excitation energies of H(2)TFcP were calculated at the TDDFT level and confirm the presence of numerous predominantly metal-to-ligand charge-transfer bands coupled via configurational interaction with expected intra-ligand pi-pi* transitions.

Mixed-valence states formation in conformationally flexible metal-free 5,10,15,20-tetraferrocenylporphyrin and 5,10-bisferrocenyl-15,20-bisphenylporphyrin.

Metal-free 5,10,15,20-tetraferrocenylporphyrin and 5,10-bisferrocenyl-15,20-bisphenylporphyrin have been prepared and characterized by UV-Vis, MCD, (1)H, (13)C, and variable-temperature NMR, APCI- and ESI-MS, and Mössbauer spectroscopy, while their redox properties were investigated using electrochemical (cyclic voltammetry and differential pulse voltammetry), spectroelectrochemical, and chemical oxidation approaches. The electronic structure calculations at Density Functional Theory level reveal that both compounds adopt saddle conformations and the HOMOs in both complexes are predominantly metal-centered, while the LUMOs predominantly consist of porphyrin pi* orbitals. In spite of the rotational freedom of ferrocenyl substituents at room temperature, both metal-free 5,10,15,20-tetraferrocenylporphyrin and 5,10-bisferrocenyl-15,20-bisphenylporphyrin are able to form mixed-valence states upon the successive ferrocene-based two- and one-electron oxidations, respectively, as confirmed by UV-Vis, MCD, Mössbauer, electro-, and spectroelectrochemical methods, and thus, the earlier suggested (Boyd et al. Chem. Commun., 1999, 637) requirements for the formation of mixed-valence states in ferrocene-containing porphyrins should be revised.