The Minimum Protein Staple? - Towards 'bio'-Baldwin's rules via inter-phosphosite linking in the MEK1 activation loop.

In small molecule organic chemistry, the heuristic insight into ring-forming processes that was enabled by Baldwin's rules some 50 years ago proved a step-change in the role of mechanistically guided synthesis. It created a lens upon and marker of fundamental stereoelectronic and conformation-guided chemical processes. However, despite the widespread role of stereoelectronics and conformational control in Biology, no equivalent coherent exploitation of trapped, ring-forming processes yet exists in biomolecules. In the development of a minimal ring-closing process in intact proteins that might prove suitable in a coherent rule-set, we have tested endo-trig ring-closing conjugate thioether lanthionine (Lan) -CH2-S-CH2- formation as a limiting cyclization. Spontaneous Lan formation in proteins is rare if not non-existent and when found in natural product cyclic peptides it requires the mediation of corresponding biosynthetic enzymes as well as productive reactive conformations to guide it. Here, we show that within a conformationally flexible and functionally important protein loop - the MAPK kinase phosphorylation-targeted activation loop - Lan ring-closing is possible. Ring-closing proves to be critically dependent on the location of a trig electrophilic site in just one of two regioisomeric potential precursors to allow phosphosite-to-phosphosite 'stapling'. This first example of spontaneous protein thioether ring-closing/'stapling' and its accessibility from just one precursor (despite the potential for both to form an identical 'staple') now reveals the potential for Lan formation not only as an accessible form of minimal stapling in proteins but also as an exquisitely sensitive probe of associated protein geometries. We suggest that the use of this (as well as the development of other such, intramolecular protein traps that are dependent on inherent protein-controlled reactivity rather than forced crosslinking) may allow the broader trapping and mapping of relevant, even minor, protein states. In this way, protein ring formation may enable a form of extended 'bio-Baldwin's rules' that help to delineate relevant protein conformational space.

Stereoretentive Post-Translational Protein Editing.

Chemical post-translational methods allow convergent side-chain editing of proteins without needing to resort to genetic intervention. Current approaches that allow the creation of constitutionally native side chains via C-C bond formation, using off-protein carbon-centered C· radicals added to unnatural amino acid radical acceptor (SOMOphile, singly occupied molecular orbital (SOMO)) "tags" such as dehydroalanine, are benign and wide-ranging. However, they also typically create epimeric mixtures of d/l-residues. Here, we describe a light-mediated desulfurative method that, through the creation and reaction of stereoretained on-proteinl-alanyl Cß· radicals, allows Cß-Hγ, Cß-Oγ, Cß-Seγ, Cß-Bγ, and Cß-Cγ bond formation to flexibly generate site-selectively edited proteins with full retention of native stereochemistry under mild conditions from a natural amino acid precursor. This methodology shows great potential to explore protein side-chain diversity and function and in the construction of useful bioconjugates.

Semicrystalline Polymer Micro/Nanostructures Formed by Droplet Evaporation of Aqueous Poly(ethylene oxide) Solutions: Effect of Solution Concentration.

Deposits formed after evaporation of sessile droplets, containing aqueous solutions of poly(ethylene oxide), on hydrophilic glass substrates were studied experimentally and mathematically as a function of the initial solution concentration. The macrostructure and micro/nanostructures of deposits were studied using stereo microscopy and atomic force microscopy. A model, based on thin-film lubrication theory, was developed to evaluate the deposit macrostructure by estimating the droplet final height. Moreover, the model was extended to evaluate the micro/nanostructure of deposits by estimating the rate of supersaturation development in connection with the driving force of crystallization. Previous studies had only described the macrostructure of poly(ethylene oxide) deposits formed after droplet evaporation, whereas the focus of our study was the deposit micro/nanostructures. Our atomic force microscopy study showed that regions close to the deposit periphery were composed of predominantly semicrystalline micro/nanostructures in the form of out-of-plane lamellae, which require a high driving force of crystallization. However, deposit central areas presented semicrystalline micro/nanostructures in the form of in-plane terraces and spirals, which require a lower driving force of crystallization. Increasing the initial concentration of solutions led to an increase in the lengths and thicknesses of the out-of-plane lamellae at the deposits' periphery and enhanced the tendency to form spirals in the central areas. Our numerical study suggested that the rate of supersaturation development and thus the driving force of crystallization increased from the center toward the periphery of droplets, and the supersaturation rate was lower for solutions with higher initial concentrations at each radius. Therefore, periphery areas of droplets with lower initial concentrations were suitable for the formation of micro/nanostructures which require higher driving forces, whereas central areas of droplets with higher initial concentration were desirable for the formation of micro/nanostructures which require lower driving forces. These numerical results were in good qualitative agreement with the experimental findings.

Persistent hypokalemia due to a rare mutation in gitelman's syndrome.

Chronic hypokalemia is the main finding in patients with Gitelman's syndrome (GS). GS, a variant of Bartter's syndrome, is an autosomal recessive renal disorder characterized by hypokalemia, hypomagnesemia, metabolic alkalosis, and hypocalciuria. GS is caused by inactivating mutations in the thiazide-sensitive sodium-chloride cotransporter gene. It is also called the "milder" form of Bartter's syndrome, as patients with GS are usually diagnosed in adulthood during routine investigation. Our objective is to highlight the impact of correct distinction between the causes of hypokalemia on management and the need of long-term follow- up after the restoration of normokalemic status. Herein, we report an asymptomatic 40-year-old male, whose persistent hypokalemia was due to GS. The diagnosis was first established by laboratory tests, and he was treated with low-dose aldosterone antagonists (spironolactone), angiotensin-converting enzyme inhibitors, and potassium and magnesium supplements. Genetic testing confirmed the diagnosis of GS and revealed a rare mutation. We conclude that GS is a rare and real diagnostic and therapeutic challenge, for which a close collaboration between endocrinologists and nephrologists is mandatory, as also the thorough genetic investigation of the mutations associated with this syndrome.

Nonisothermal Spreading Dynamics of Self-Rewetting Droplets.

We experimentally studied the spreading dynamics of binary alcohol mixtures (and pure liquids for reference) deposited on a heated substrate in a partially wetting situation under nonisothermal conditions. We show that the spreading mechanism of an evaporating droplet exhibits a power-law growth with early-stage exponents that depend strongly and nonmonotonically on the substrate temperature. Moreover, we investigated the temporal and spatial thermal dynamics in the droplet using infrared thermography, revealing the existence of unique thermal patterns due to thermal and/or solutal instabilities, which lead to surface tension gradients, namely the Marangoni effect. Our key findings are that the temperature of the substrate drastically affects the early-stage inertial-capillary spreading regime owing to the nonmonotonic surface tension-temperature dependence of the self-rewetting liquids. At later stages of wetting, the spreading dynamics enters the viscous-capillary dominated regime, with the characteristic low kinetics mirroring the behavior of pure liquids.

Effect of Poly(ethylene oxide) Molecular Weight on the Pinning and Pillar Formation of Evaporating Sessile Droplets: The Role of the Interface.

We report on the drying process of sessile droplets of aqueous poly(ethylene oxide) (PEO) solutions studied by contact angle analysis. Liquid samples were prepared with the same initial concentration of four different molecular weights, Mw, of PEO. Droplets with initial volumes of between 1 and 5 µL were left to evaporate while temperature, pressure, and relative humidity were kept constant. Residues were formed with either a disklike puddle or a distinctive tall conical pillar shape. The latter occurred following a four-stage deposition process: pinned drying, during which the contact line is stationary; pseudodewetting, where the receding contact line is induced by precipitation; bootstrap building, during which the liquid droplet is lifted on freshly precipitated solid; and late drying. Contact angle analysis allowed us to monitor all stages during drying and consider transitions between stages for different molecular weights. We illustrate the mechanisms taking place during the crucial stages of pinning and depinning, revealing the effect of adhesion and contact line friction for high molecular weights and its influence on the final morphology of the dried PEO solute. To this end, we performed PEO solution droplet evaporation on PEO and PTFE films demonstrating the importance of interfacial interaction phenomena. We show that the formation of disklike puddles for high molecular weights on glass is associated with continuous droplet contact line pinning. This results from the strong adhesion due to the interdigitation of the loops and tails of a polymer layer (adsorbed on glass during evaporation) with the polymer gel network inside the droplet that forms as water evaporates.