Sometimes less is more: [13C1H3]methyl isotopomers can be biosynthetically incorporated specifically into the pro-S methyl groups of leucine and valine residues in large protein assemblies within a perdeuterated background by using an acetolactate precursor. This stereospecific labeling strategy considerably enhances NMR spectra for assemblies. Detailed facts importance to specialist readers are published as ”Supporting Information”. Such documents peer-reviewed, but not copy-edited or...

While biophysical studies have unravelled properties of specific proteins in vitro , characterizing globally their native state within the cell remains a challenge. In particular, protein adaptation to harsh intracellular physical and chemical conditions is poorly understood. Extremophiles, which thrive severe environments, are good models for study such adaptation. Five haloarchaeal species, isolated from hypersaline were used assess correlations between salt concentrations molecular...

Intracellular water dynamics in Haloarcula marismortui , an extremely halophilic organism originally isolated from the Dead Sea, was studied by neutron scattering. The centrifuged cell pellets examined means of two spectrometers, IN6 and IN16, sensitive to motions with time scales 10 ps 1 ns, respectively. From data, a translational diffusion constant 1.3 × −5 cm 2 s −1 determined at 285 K. This value is close that found previously for other cells bulk water, as well 3.5 M NaCl solution...

An optimized NMR experiment that combines the advantages of methyl-TROSY and SOFAST-HMQC has been developed. It allows recording high quality methyl 1H−13C correlation spectra protein assemblies several hundreds kDa in a few seconds. The SOFAST-methyl-TROSY-based offers completely new opportunities for study structural dynamic changes occurring molecular nanomachines while they perform their biological function vitro.

Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus closely related pressure-sensitive kodakarensis, at 0.1 40 MPa. We show that cells of pressure sensitive organism exhibit higher intrinsic stability. Both hydration water fast lipid are reduced under pressure. In contrast, proteome T. is more than kodakarensis. The diffusion coefficient reduced, while slightly enhanced with increasing These...

To explore protein adaptation to extremely high temperatures, two parameters related macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii mesophilic homologue, lactate Oryctolagus cunniculus (rabbit) muscle. The root fluctuations, defining flexibility, found be similar both enzymes (1.5 Å) at their optimal activity...

Abstract Halophilic proteins subjected to below about 15% salt in vitro denature through misfolding, aggregation and/or precipitation. Halobacteria, however, have been detected environments of fluctuating salinity such as coastal salterns and even around fresh water springs the depths Dead Sea. In order identify underlying mechanisms low survival, we explored reactivation capacity Halobacterium (Hbt) salinarum sub-populations after incubation media recovery physiological salt. Respiratory...

Obtaining crystals and solving the phase problem remain major hurdles encountered by bio-crystallographers in their race to obtain new high-quality structures. Both issues can be overcome crystallophore, Tb-Xo4, a lanthanide-based molecular complex with unique nucleating phasing properties. This article presents examples of crystallization conditions induced presence Tb-Xo4. These crystalline forms bypass crystal defects often crystallographers, such as low-resolution diffracting samples or...

M42 peptidases are half-megadalton aminopeptidases characterized by a tetrahedral architecture (TET) ubiquitous across all domains of life. Despite their widespread occurrence, evolutionary history and functional diversity remain largely unexplored. Here we show an unsuspected untapped wealth archaeal TET peptidases, exhibiting remarkable heterogeneity, as illustrated the characterization six novel enzymes. Using structural biology, phylogeny, enzymatic studies, establish robust criteria for...

We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation fluorescence spectroscopy as novel approach to obtain time-resolved, structural data individually from macromolecular complexes their substrates during active biochemical reactions. The allowed us monitor the mechanical unfolding green fluorescent protein model substrate by archaeal AAA+ PAN unfoldase on sub-minute time scale. Concomitant with its substrate, complex...

Abstract The majority of the biosphere is a high pressure environment. Around 70% marine lies at depths below 1000 m, i.e. pressures 100 bars or higher. To survive in these environments, deep-biosphere organisms have adapted to life pressure. In vitro studies showed that activity certain proteins originating from deep-sea less affected by than enzymes surface . However, genetic and structural bases for this increased resistance are still unknown. Elastic incoherent neutron scattering...

Glyoxylate accumulation within cells is highly toxic. In humans, it associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are conserved enzymes a dual activity as they able reduce glycolate and convert hydroxypyruvate into D-glycerate. Despite determination of high-resolution X-ray structures, substrate recognition mode this class remains unclear. We determined...

Control of translation is an important step in chloroplast gene expression. A first control can be exerted during the initiation complex formation which, Escherichia coli, involves ribosomal protein (r-protein) S1. cDNA clone have been characterized which codes for precursor r-protein CS1. The mature consists a central core shows 31.5% amino acid homology to E. coli CS1 considerably shorter (40 kDa) than S1 (61 kDa). fragment contains three degenerated repeats show both ribosome- and...

The structure of a 468 kDa peptidase complex from the hyperthermophile Pyrococcus horikoshii has been solved at 1.9 A resolution. monomer contains M42 typical catalytic domain, and dimerization domain that allows formation dimers assemble as 12-subunit self-compartmentalized tetrahedron, similar to those described for TET peptidases. biochemical analysis shows enzyme is cobalt-activated cleaves peptides by non-processive mechanism. Consequently, this protein represents third in P....