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White-Light-Emitting Lanthanide Metallogels with Tunable Luminescence and Reversible Stimuli-Responsive Properties
Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro
Oxalates as Activating Groups for Alcohols in Visible Light Photoredox Catalysis: Formation of Quaternary Centers by Redox-Neutral Fragment Coupling
Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging
Facile Phase Transfer and Surface Biofunctionalization of Hydrophobic Nanoparticles Using Janus DNA Tetrahedron Nanostructures
Hfqs in Bacillus anthracis: Role of protein sequence variation in the structure and function of proteins in the Hfq family
Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in Escherichia coli, was characterized. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer–hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation.
Intracellular pH modulates quinary structure
NMR spectroscopy can provide information about proteins in living cells. pH is an important characteristic of the intracellular environment because it modulates key protein properties such as net charge and stability. Here, we show that pH modulates quinary interactions, the weak, ubiquitous interactions between proteins and other cellular macromolecules. We use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells. By controlling the intracellular pH, we show that quinary interactions influence the quality of in-cell 15N–1H HSQC NMR spectra. At low pH, the quality is degraded because the increase in attractive interactions between E. coli proteins and GB1 slows GB1 tumbling and broadens its crosspeaks. The results demonstrate the importance of quinary interactions for furthering our understanding of protein chemistry in living cells.
Assessment of the utility of contact-based restraints in accelerating the prediction of protein structure using molecular dynamics simulations
Molecular dynamics (MD) simulation is a well-established tool for the computational study of protein structure and dynamics, but its application to the important problem of protein structure prediction remains challenging, in part because extremely long timescales can be required to reach the native structure. Here, we examine the extent to which the use of low-resolution information in the form of residue–residue contacts, which can often be inferred from bioinformatics or experimental studies, can accelerate the determination of protein structure in simulation. We incorporated sets of 62, 31, or 15 contact-based restraints in MD simulations of ubiquitin, a benchmark system known to fold to the native state on the millisecond timescale in unrestrained simulations. One-third of the restrained simulations folded to the native state within a few tens of microseconds—a speedup of over an order of magnitude compared with unrestrained simulations and a demonstration of the potential for limited amounts of structural information to accelerate structure determination. Almost all of the remaining ubiquitin simulations reached near-native conformations within a few tens of microseconds, but remained trapped there, apparently due to the restraints. We discuss potential methodological improvements that would facilitate escape from these near-native traps and allow more simulations to quickly reach the native state. Finally, using a target from the Critical Assessment of protein Structure Prediction (CASP) experiment, we show that distance restraints can improve simulation accuracy: In our simulations, restraints stabilized the native state of the protein, enabling a reasonable structural model to be inferred.
Highly Efficient Dual-Color Electrochemiluminescence from BODIPY-Capped PbS Nanocrystals
Synthesis of Adjacent Quaternary Stereocenters by Catalytic Asymmetric Allylboration
Seed-Mediated Growth of Anatase TiO2 Nanocrystals with Core–Antenna Structures for Enhanced Photocatalytic Activity
Functional footprinting of regulatory DNA
Nature Methods 12, 927 (2015). doi:10.1038/nmeth.3554
Authors: Jeff Vierstra, Andreas Reik, Kai-Hsin Chang, Sandra Stehling-Sun, Yuanyue Zhou, Sarah J Hinkley, David E Paschon, Lei Zhang, Nikoletta Psatha, Yuri R Bendana, Colleen M O'Neil, Alexander H Song, Andrea K Mich, Pei-Qi Liu, Gary Lee, Daniel E Bauer, Michael C Holmes, Stuart H Orkin, Thalia Papayannopoulou, George Stamatoyannopoulos, Edward J Rebar, Philip D Gregory, Fyodor D Urnov & John A Stamatoyannopoulos
Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing–induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.
Inntags: small self-structured epitopes for innocuous protein tagging
Nature Methods 12, 955 (2015). doi:10.1038/nmeth.3556
Authors: Maya V Georgieva, Galal Yahya, Laia Codó, Raúl Ortiz, Laura Teixidó, José Claros, Ricardo Jara, Mònica Jara, Antoni Iborra, Josep Lluís Gelpí, Carme Gallego, Modesto Orozco & Martí Aldea
Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. However, an intrinsic limitation of tagging is that the native function of the protein may be compromised or even abolished by the presence of the tag. Here we describe and characterize a set of small, innocuous protein tags (inntags) that we anticipate will find application in a variety of biological techniques.
Programmed synthesis of three-dimensional tissues
Nature Methods 12, 975 (2015). doi:10.1038/nmeth.3553
Authors: Michael E Todhunter, Noel Y Jee, Alex J Hughes, Maxwell C Coyle, Alec Cerchiari, Justin Farlow, James C Garbe, Mark A LaBarge, Tejal A Desai & Zev J Gartner
CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo
Nature Methods 12, 982 (2015). doi:10.1038/nmeth.3543
Authors: Miguel A Moreno-Mateos, Charles E Vejnar, Jean-Denis Beaudoin, Juan P Fernandez, Emily K Mis, Mustafa K Khokha & Antonio J Giraldez
Cochlear hair cells: The sound-sensing machines
