SNAP-tag2 for faster and brighter protein labeling.

SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here, we introduce improved labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells.

Two-Photon-Driven Photoprotection Mechanism in Echinenone-Functionalized Orange Carotenoid Protein.

Orange carotenoid protein (OCP) is a photoactive protein that mediates photoprotection in cyanobacteria. OCP binds different ketocarotenoid chromophores such as echinenone (ECN), 3'- hydroxyechinenone (hECN), and canthaxanthin (CAN). In the dark, OCP is in an inactive orange form known as OCP; upon illumination, a red active state is formed, referred to as OCP, that can interact with the phycobilisome.

Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag.

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates.

The Trimeric Major Capsid Protein of Mavirus is stabilized by its Interlocked N-termini Enabling Core Flexibility for Capsid Assembly.

Icosahedral viral capsids assemble with high fidelity from a large number of identical buildings blocks. The mechanisms that enable individual capsid proteins to form stable oligomeric units (capsomers) while affording structural adaptability required for further assembly into capsids are mostly unknown. Understanding these mechanisms requires knowledge of the capsomers' dynamics, especially for viruses where no additional helper proteins are needed during capsid assembly like for the Mavirus virophage that despite its complexity (triangulation number T = 27) can assemble from its major capsid protein (MCP) alone.

Multiquantum Chemical Exchange Saturation Transfer NMR to Quantify Symmetrical Exchange: Application to Rotational Dynamics of the Guanidinium Group in Arginine Side Chains.

Chemical exchange saturation transfer (CEST) NMR experiments have emerged as a powerful tool for characterizing dynamics in proteins. We show here that the CEST approach can be extended to systems with symmetrical exchange, where the NMR signals of all exchanging species are severely broadened. To achieve this, multiquantum CEST (MQ-CEST) is introduced, where the CEST pulse is applied to a longitudinal multispin order density element and the CEST profiles are encoded onto nonbroadened nuclei.

Illumination guidelines for ultrafast pump-probe experiments by serial femtosecond crystallography.

Time-resolved crystallography with X-ray free-electron lasers enables structural characterization of light-induced reactions on ultrafast timescales. To be biologically and chemically relevant, such studies must be carried out in an appropriate photoexcitation regime to avoid multiphoton artifacts, a common issue in recent studies. We describe numerical and experimental approaches to determine how many photons are needed for single-photon excitation in microcrystals, taking into account losses by scattering.

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin.

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds.

Capsid protein structure, self-assembly, and processing reveal morphogenesis of the marine virophage mavirus.

Virophages have the unique property of parasitizing giant viruses within unicellular hosts. Little is understood about how they form infectious virions in this tripartite interplay. We provide mechanistic insights into assembly and maturation of mavirus, a marine virophage, by combining structural and stability studies on capsomers, virus-like particles (VLPs), and native virions.

Bap (Sil1) regulates the molecular chaperone BiP by coupling release of nucleotide and substrate.

BiP is the endoplasmic member of the Hsp70 family. BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) Bap (Sil1 in yeast). Bap is a two-domain protein.

Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl Cyclase.

Light-regulated enzymes enable organisms to quickly respond to changing light conditions. We characterize a photoactivatable adenylyl cyclase (AC) from Beggiatoa sp. (bPAC) that translates a blue light signal into the production of the second messenger cyclic AMP.

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

Regulation of the P450 Oxygenation Cascade Involved in Glycopeptide Antibiotic Biosynthesis.

Glycopeptide antibiotics (GPAs) are nonribosomal peptides rich in modifications introduced by external enzymes. These enzymes act on the free peptide aglycone or intermediates bound to the nonribosomal peptide synthetase (NRPS) assembly line. In this process the terminal module of the NRPS plays a crucial role as it contains a unique recruitment platform (X-domain) interacting with three to four modifying Cytochrome P450 (P450) enzymes that are responsible for cyclizing bound peptides.

Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation.

The hemoprotein myoglobin is a model system for the study of protein dynamics. We used time-resolved serial femtosecond crystallography at an x-ray free-electron laser to resolve the ultrafast structural changes in the carbonmonoxy myoglobin complex upon photolysis of the Fe-CO bond. Structural changes appear throughout the protein within 500 femtoseconds, with the C, F, and H helices moving away from the heme cofactor and the E and A helices moving toward it.

Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1.

RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1's enzymatic function is unknown.

trans-Acting arginine residues in the AAA+ chaperone ClpB allosterically regulate the activity through inter- and intradomain communication.

The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine. Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which also harbor the catalytic sites.

In vitro analysis of human immunodeficiency virus particle dissociation: gag proteolytic processing influences dissociation kinetics.

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice.

Hexicon 2: automated processing of hydrogen-deuterium exchange mass spectrometry data with improved deuteration distribution estimation.

Hydrogen-deuterium exchange (HDX) experiments analyzed by mass spectrometry (MS) provide information about the dynamics and the solvent accessibility of protein backbone amide hydrogen atoms. Continuous improvement of MS instrumentation has contributed to the increasing popularity of this method; however, comprehensive automated data analysis is only beginning to mature. We present Hexicon 2, an automated pipeline for data analysis and visualization based on the previously published program Hexicon (Lou et al.

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor.

ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically.

Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.

A variety of enzymes are activated by the binding of potassium ions. The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. In these cases, ammonium can be used as a proxy for potassium to characterise potassium binding sites in enzymes: the (1) H,(15) N spin-pair of enzyme-bound (15) NH4 (+) can be probed by (15) N-edited heteronuclear NMR experiments.

Characterization of elements involved in allosteric light regulation of phosphodiesterase activity by comparison of different functional BlrP1 states.

Bacteria have evolved dedicated signaling mechanisms that enable the integration of a range of environmental stimuli and the accordant modulation of metabolic pathways. One central signaling molecule in bacteria is the second messenger cyclic dimeric GMP (c-di-GMP). Complex regulatory mechanisms for modulating c-di-GMP concentrations have evolved, in line with its importance for maintaining bacterial fitness under changing environmental conditions.

Folding properties of cytosine monophosphate kinase from E. coli indicate stabilization through an additional insert in the NMP binding domain.

The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ.

Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own.

Structural Insights into the Effector - Immunity System Tae4/Tai4 from Salmonella typhimurium.

Type-6-secretion systems of Gram-negative bacteria are widely distributed needle-like multi-protein complexes that are involved in microbial defense mechanisms. During bacterial competition these injection needles dispense effector proteins into the periplasm of competing bacteria where they induce degradation of the peptidoglycan scaffold and lead to cell lysis. Donor cells co-produce immunity proteins and shuttle them into their own periplasm to prevent accidental toxication by siblings.

A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression.

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions.