SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.
Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded -barrel with a PWWP-specific long loop connecting 2 and 3 (PR-loop), followed by a helical region including two ␣-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the 1/2 loop (- arch) and the 3/4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.
By using in-cell NMR experiments, we have demonstrated that the protein folding state in cells is significantly influenced by the cellular health conditions. hAK1 was denatured in cells under stressful culture conditions, while it remained functional and properly folded in cells continuously supplied with a fresh medium.
Intracellular cargo delivery plays an important role in fundamental biological research [1] and therapeutic medical applications, [2] ranging from intracellular function analysis, [3] gene encoding for cellular reprogramming, [4] and the inhibition of gene expression inside cells. [5] Cargo delivery requires safe and efficient access to cells and different intracellular locations such as the nucleus and mitochondria due to impermeable outer cell membranes, including small molecules, [6] nucleic acid genes, [7] amino acid proteins, [8] nanosensors, [9] and organelles. [10] Nanostructures are promising candidates for membrane disruption where versatile cargos transport into cells to overcome the impermeable plasma membrane. Silicon nanowires, [11] diamond nanoneedles, [12] carbon nanofibers, [13] and ZnO nanowires [14] have been developed for mechanical needle penetration into living cells for molecular delivery. Although they improved the delivery efficiency, but the cargo delivery is still restricted by some limitations such as small amount of the loaded/released molecules to/from the nanostructure surfaces (dosage/dosage control). Consequently, hollow nanostructures, including silicon, [15] carbon, [16] and Al 2 O 3 [17] nanotubes (NTs), spontaneously penetrated cells with connection to microfluidic channels, which can control molecular flow and subsequent direct delivery into cells through nanostructured ducts. A plasmid DNA, [18] Ca 2þ indicator, [19] fluorescent dye, [20] quantum dot, [21] and protein [22] were delivered into different cell types using the aforementioned methods. For further improvement, the NTs were combined with external forces such as mechanical, [18] electrical, [23] and photothermal poration. [24,25] Among these methods, an NTelectroporation platform is an excellent technique for intracellular delivery, providing improvement in delivery efficiency and dosage controllability. However, the cell viability still remained a problem due to the high voltages requirement of over 1.5 V to create transient pores in the plasma membrane. [26] Furthermore, such high electrical voltage induces problematic intracellular signaling, which is relative to differentiation [27] and reprogramming. [28] Here, we develop metal-organic hybrid NTs that can be inserted into adhesive cells and subsequently deliver versatile
Ras acts as a molecular switch to control intracellular signaling on the plasma membrane (PM). Elucidating how Ras associates with PM in the native cellular environment is crucial for understanding its control mechanism. Here, we used in-cell nuclear magnetic resonance (NMR) spectroscopy combined with site-specific 19 F-labeling to explore the membrane-associated states of H-Ras in living cells. The site-specific incorporation of p-trifluoromethoxyphenylalanine (OCF 3 Phe) at three different sites of H-Ras, i.e., Tyr32 in switch I, Tyr96 interacting with switch II, and Tyr157 on helix α5, allowed the characterization of their conformational states depending on the nucleotide-bound states and an oncogenic mutational state. Exogenously delivered 19 F-labeled H-Ras protein containing a Cterminal hypervariable region was assimilated via endogenous membrane-trafficking, enabling proper association with the cell membrane compartments. Despite poor sensitivity of the in-cell NMR spectra of membrane-associated H-Ras, the Bayesian spectral deconvolution identified distinct signal components on three 19 F-labeled sites, thus offering the conformational multiplicity of H-Ras on the PM. Our study may be helpful in elucidating the atomic-scale picture of membrane-associated proteins in living cells.
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