C 2 -domains are widespread protein modules with diverse Ca 2⍣ -regulatory functions. Although multiple Ca 2⍣ ions are known to bind at the tip of several C 2 -domains, the exact number of Ca 2⍣ -binding sites and their functional relevance are unknown. The first C 2 -domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca 2⍣ -dependent interactions with syntaxin and phospholipids. We have studied the Ca 2⍣ -binding mode of this C 2 -domain as a prototypical C 2 -domain using NMR spectroscopy and site-directed mutagenesis. The C 2 -domain is an elliptical module composed of a β-sandwich with a long axis of 50 Å. Our results reveal that the C 2 -domain binds three Ca 2⍣ ions in a tight cluster spanning only 6 Å at the tip of the module. The Ca 2⍣ -binding region is formed by two loops whose conformation is stabilized by Ca 2ϩ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C 2 -domains. All three Ca 2⍣ ions are required for the interactions of the C 2 -domain with syntaxin and phospholipids. These results support an electrostatic switch model for C 2 -domain function whereby the β-sheets of the domain provide a fixed scaffold for the Ca 2⍣ -binding loops, and whereby interactions with target molecules are triggered by a Ca 2⍣ -induced switch in electrostatic potential.

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.

Upon fertilization, drastic chromatin reorganization occurs during preimplantation development . However, the global chromatin landscape and its molecular dynamics in this period remain largely unexplored in humans. Here we investigate chromatin states in human preimplantation development using an improved assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) . We find widespread accessible chromatin regions in early human embryos that overlap extensively with putative cis-regulatory sequences and transposable elements. Integrative analyses show both conservation and divergence in regulatory circuitry between human and mouse early development, and between human pluripotency in vivo and human embryonic stem cells. In addition, we find widespread open chromatin regions before zygotic genome activation (ZGA). The accessible chromatin loci are readily found at CpG-rich promoters. Unexpectedly, many others reside in distal regions that overlap with DNA hypomethylated domains in human oocytes and are enriched for transcription factor-binding sites. A large portion of these regions then become inaccessible after ZGA in a transcription-dependent manner. Notably, such extensive chromatin reorganization during ZGA is conserved in mice and correlates with the reprogramming of the non-canonical histone mark H3K4me3, which is uniquely linked to genome silencing. Taken together, these data not only reveal a conserved principle that underlies the chromatin transition during mammalian ZGA, but also help to advance our understanding of epigenetic reprogramming during human early development and in vitro fertilization.

Synaptotagmin I is a synaptic vesicle protein that is thought to act as a Ca2+ sensor in neurotransmitter release. The first C2 domain of synaptotagmin I (C2A domain) contains a bipartite Ca2+-binding motif and interacts in a Ca2+-dependent manner with syntaxin, a central component of the membrane fusion complex. Analysis by nuclear magnetic resonance spectroscopy and site-directed mutagenesis shows that this interaction is mediated by the cooperative action of basic residues surrounding the Ca2+-binding sites of the C2A domain and is driven by a change in the electrostatic potential of the C2A domain induced by Ca2+ binding. A model is proposed whereby synaptotagmin acts as an electrostatic switch in Ca2+-triggered synaptic vesicle exocytosis, promoting a structural rearrangement in the fusion machinery that is effected by its interaction with syntaxin.

Synaptotagmin I is a synaptic vesicle membrane protein that probably functions as a Ca2+ sensor in neurotransmitter release and contains two C2-domains which bind Ca2+. The first C2-domain of synaptotagmin I (the C2A-domain) binds phospholipids in a Ca2+-dependent manner similar to that of the C2-domains of protein kinase C, cytoplasmic phospholipase A2, and phospholipase Cdelta1. Although the tertiary structure of these C2-domains is known, the molecular basis for their Ca2+-dependent interactions with phospholipids is unclear. We have now investigated the mechanisms involved in Ca2+-dependent phospholipid binding by the C2A-domain of synaptotagmin I. Our data show that the C2A-domain binds negatively charged liposomes in an electrostatic interaction that is determined by the charge density of the liposome surface but not by the phospholipid headgroup. At the tip of the C2A-domain, three tightly clustered Ca2+-binding sites are formed by five aspartates and one serine. Mutations in these aspartate and serine residues demonstrated that all three Ca2+-binding sites are required for phospholipid binding. The Ca2+ binding sites at the top of the C2A-domain are surrounded by positively charged amino acids that were shown by mutagenesis to be also involved in phospholipid binding. Our results yield a molecular picture of the interactions between a C2-domain and phospholipids. Binding is highly electrostatic and occurs between the surfaces of the phospholipid bilayer and of the tip of the C2A-domain. The data suggest that the negatively charged phospholipid headgroups interact with the basic side chains surrounding the Ca2+-binding sites and with bound Ca2+ ions, thereby filling empty coordination sites and increasing the apparent affinity for Ca2+. In addition, insertion of hydrophobic side chains may contribute to phospholipid binding. This model is likely to be general for other C2-domains, with the relative contributions of electrostatic and hydrophobic interactions dictated by the exposed side chains surrounding the Ca2+-binding region.

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Highlights Psychological disturbances of frontline medical staff are more than those of general population. Daily working hours are a risk factor for all measured psychological disturbances in frontline medical staff. Some other factors may be involved in certain psychological disturbances of frontline medical staff.

A novel fluorescent probe with n,π* configuration, the azoalkane 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO), is responsive to complexation by supramolecular hosts. The n,π* fluorescent probe serves to provide structural and, owing to its exceedingly long fluorescence lifetime (up to 1 μs), also kinetic information on host−guest complexation. The three cyclodextrins (CDs) were selected as prototypal hosts in aqueous solution, and the complexation, in both the ground and excited states, was followed by four techniques:  time-resolved and steady-state fluorescence, UV absorption spectrophotometry, and NMR spectroscopy. The fluorescence quenching rate constants (k q) of DBO by α-, β-, and γ-CD (1.9, 4.0, and 0.78 × 108 M-1 s-1) were determined from the dynamic component of the biexponential time-resolved decay traces, while the static component was assigned to the fluorescence lifetimes of the complexes (τ CD) of α-CD (ca. 33 ns) and β-CD (ca. 95 ns). Time-resolved and steady-state fluorescence measurements yielded consistent results. The shorter lifetimes in the complexes are attributed to the propensity of singlet-excited DBO to undergo fluorescence quenching by an “aborted” hydrogen abstraction with the labile glycosidic C−H bonds inside the cavity. Ground-state binding constants (K) could be determined by both UV spectrophotometry (for β-CD, ca. 900 M-1) and, owing to the high water solubility, also by NMR spectroscopy to afford values of 50, 1100, and 6 M-1 for α-, β-, and γ-CD, respectively. The spectroscopic data support the formation of inclusion complexes in both the excited and ground states. The dynamic quenching is attributed to inclusion with subsequent quenching inside the shorter-lived complex. The examination of the complexation dynamics at high guest (DBO) concentration revealed an unprecedented behavior, which may be indicative of singlet energy transfer between the free DBO and the CD·DBO complex. The potential of DBO as a distinct and complementary fluorescent probe is discussed.