An integrative characterization of proline cis and trans conformers in a disordered peptide.

Intrinsically disordered proteins (IDPs) often contain proline residues that undergo cis/trans isomerization. While molecular dynamics (MD) simulations have the potential to fully characterize the proline cis and trans subensembles, they are limited by the slow timescales of isomerization and force field inaccuracies. NMR spectroscopy can report on ensemble-averaged observables for both the cis-proline and trans-proline states, but a full atomistic characterization of these conformers is challenging.

The ribosome lowers the entropic penalty of protein folding.

Most proteins fold during biosynthesis on the ribosome, and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies. The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation.

The Influence of Disulfide, Thioacetal and Lanthionine-Bridges on the Conformation of a Macrocyclic Peptide.

Cyclisation of peptides by forming thioether (lanthionine), disulfide (cystine) or methylene thioacetal bridges between side chains is established as an important tool to stabilise a given structure, enhance metabolic stability and optimise both potency and selectivity. However, a systematic comparative study of the effects of differing bridging modalities on peptide conformation has not previously been carried out. In this paper, we have used the NMR deconvolution algorithm, NAMFIS, to determine the conformational ensembles, in aqueous solution, of three cyclic analogues of angiotensin(1-7), incorporating either disulfide, or non-reducible thioether or methylene thioacetal bridges.

Picosecond Dynamics of a Small Molecule in Its Bound State with an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules.

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers.

m6A methylation provides an essential layer of regulation in organismal development, and is aberrant in a range of cancers and neuro-pathologies. The information encoded by m6A methylation is integrated into existing RNA regulatory networks by RNA binding proteins that recognise methylated sites, the m6A readers. m6A readers include a well-characterised class of dedicated proteins, the YTH proteins, as well as a broader group of multi-functional regulators where recognition of m6A is only partially understood.

Molecular Recognition of Lipid II by Lantibiotics: Synthesis and Conformational Studies of Analogues of Nisin and Mutacin Rings A and B.

In response to the growing threat posed by antibiotic-resistant bacterial strains, extensive research is currently focused on developing antimicrobial agents that target lipid II, a vital precursor in the biosynthesis of bacterial cell walls. The lantibiotic nisin and related peptides display unique and highly selective binding to lipid II. A key feature of the nisin-lipid II interaction is the formation of a cage-like complex between the pyrophosphate moiety of lipid II and the two thioether-bridged rings, rings A and B, at the N-terminus of nisin.

Structural and functional adaptation of Haloferax volcanii TFEα/β.

The basal transcription factor TFE enhances transcription initiation by catalysing DNA strand-separation, a process that varies with temperature and ionic strength. Canonical TFE forms a heterodimeric complex whose integrity and function critically relies on a cubane iron-sulphur cluster residing in the TFEβ subunit. Halophilic archaea such as Haloferax volcanii have highly divergent putative TFEβ homologues with unknown properties.

Determining rotational dynamics of the guanidino group of arginine side chains in proteins by carbon-detected NMR.

Arginine residues are imperative for many active sites and protein-interaction interfaces. A new NMR-based method is presented to determine the rotational dynamics around the N-C bond of arginine side chains. An application to a 19 kDa protein shows that the strengths of interactions involving arginine side chains can be characterised.

Epitope mapping of imidazolium cations in ionic liquid-protein interactions unveils the balance between hydrophobicity and electrostatics towards protein destabilisation.

We investigated imidazolium-based ionic liquid (IL) interactions with human serum albumin (HSA) to discern the level of cation interactions towards protein stability. STD-NMR spectroscopy was used to observe the imidazolium IL protons involved in direct binding and to identify the interactions responsible for changes in Tm as accessed by differential scanning calorimetry (DSC). Cations influence protein stability less than anions but still significantly.

Protein destabilisation in ionic liquids: the role of preferential interactions in denaturation.

The preferential binding of anions and cations in aqueous solutions of the ionic liquids (ILs) 1-butyl-3-methylimidazolium ([C4mim](+)) and 1-ethyl-3-methylimidazolium ([C2mim](+)) chloride and dicyanamide (dca(-)) with the small alpha-helical protein Im7 was investigated using a combination of differential scanning calorimetry, NMR spectroscopy and molecular dynamics (MD) simulations. Our results show that direct ion interactions are crucial to understand the effects of ILs on the stability of proteins and that an anion effect is dominant. We show that the binding of weakly hydrated anions to positively charged or polar residues leads to the partial dehydration of the backbone groups, and is critical to control stability, explaining why dca(-) is more denaturing than Cl(-).

Conformational dynamics is more important than helical propensity for the folding of the all α-helical protein Im7.

Im7 folds via an on-pathway intermediate that contains three of the four native α-helices. The missing helix, helix III, is the shortest and its failure to be formed until late in the pathway is related to frustration in the structure. Im7H3M3, a 94-residue variant of the 87-residue Im7 in which helix III is the longest of the four native helices, also folds via an intermediate.

Effects of transaldolase exchange on estimates of gluconeogenesis in type 2 diabetes.

Transaldolase (TA) exchange overestimates gluconeogenesis measured with deuterated water (²H₂O). However, it is unknown whether TA differs in people with type 2 diabetes (T2DM). ²H₂O was ingested, and [1-¹³C]acetate and [3-³H]glucose were infused in T2DM (n = 10) and healthy nondiabetic (ND; n = 8) subjects.

Understanding how small helical proteins fold: conformational dynamics of Im proteins relevant to their folding landscapes.

Understanding the mechanism of folding of small proteins requires characterization of their starting unfolded states and any partially unfolded states populated during folding. Here, we review what is known from NMR about these states of Im7, a 4-helix bundle protein that folds via an on-pathway intermediate, and show that there is an alignment of non-native structure in urea-unfolded Im7 with the helices of native Im7 that is a consequence of hydrophobic helix-promoting residues also promoting cluster-formation in the unfolded protein. We suggest that this kind of alignment is present in other proteins and is relevant to how native state topology determines folding rates.

Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7.

The small (87-residue) alpha-helical protein Im7 (an inhibitor protein for colicin E7 that provides immunity to cells producing colicin E7) folds via a three-state mechanism involving an on-pathway intermediate. This kinetic intermediate contains three of four native helices that are oriented in a non-native manner so as to minimise exposed hydrophobic surface area at this point in folding. The short (6-residue) helix III has been shown to be unstructured in the intermediate ensemble and does not dock onto the developing hydrophobic core until after the rate-limiting transition state has been traversed.

Purification and preliminary characterization of tetraheme cytochrome c3 and adenylylsulfate reductase from the peptidolytic sulfate-reducing bacterium Desulfovibrio aminophilus DSM 12254.

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns).