Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein

De-novo designed proteins have received wide interest as potential platforms for nano-engineering and biomedicine. While much work is being done in the design of thermodynamically stable proteins, the folding process of artificially designed proteins is not well-studied. Here we used single-molecule force spectroscopy by optical tweezers to study the folding of ROSS, a de-novo designed 2x2 Rossmann fold. We measured a barrier crossing time in the millisecond range, much slower than what has been reported for other systems. While long transition times can be explained by barrier roughness or slow diffusion, we show that isotropic roughness cannot explain the measured transition path time distribution. Instead, this study shows that the slow barrier crossing of ROSS is caused by the population of three short-lived high-energy intermediates. In addition, we identify incomplete and off-pathway folding events with different barrier crossing dynamics. Our results hint at the presence of a complex transition barrier that may be a common feature of many artificially designed proteins

Auto- and cross-power spectral analysis of dual trap optical tweezer experiments using Bayesian inference

The thermal fluctuations of micron-sized beads in dual trap optical tweezer experiments contain complete dynamic information about the viscoelastic properties of the embedding medium and—if present—macromolecular constructs connecting the two beads. To quantitatively interpret the spectral properties of the measured signals, a detailed understanding of the instrumental characteristics is required. To this end, we present a theoretical description of the signal processing in a typical dual trap optical tweezer experiment accounting for polarization crosstalk and instrumental noise and discuss the effect of finite statistics. To infer the unknown parameters from experimental data, a maximum likelihood method based on the statistical properties of the stochastic signals is derived. In a first step, the method can be used for calibration purposes: We propose a scheme involving three consecutive measurements (both traps empty, first one occupied and second empty, and vice versa), by which all instrumental and physical parameters of the setup are determined. We test our approach for a simple model system, namely a pair of unconnected, but hydrodynamically interacting spheres. The comparison to theoretical predictions based on instantaneous as well as retarded hydrodynamics emphasizes the importance of hydrodynamic retardation effects due to vorticity diffusion in the fluid. For more complex experimental scenarios, where macromolecular constructs are tethered between the two beads, the same maximum likelihood method in conjunction with dynamic deconvolution theory will in a second step allow one to determine the viscoelastic properties of the tethered element connecting the two beads

Ecological implications of pedogenesis and geochemistry of ultramafic soils in Kinabalu Park (Malaysia)

In Sabah, Malaysia, ultramafic rock outcrops are widespread (totalling 3500 km2, one of the main outcrops in the tropical zone), and predominantly of the peridotite type. However, strongly serpentinised peridotite is also locally common, particularly along fault lines in the Mt. Kinabalu area. This study aimed to determine the extent of chemical variation in ultramafic soils in relation to the degree of serpentinisation and the weathering intensity, and consequent potential ecological implications linked to resulting soil chemical fertility. It was hypothesized that young soils and soils derived from bedrock with a significant degree of serpentinisation strongly differ from typical Geric Ferralsols and result in soil chemistries with more adverse properties to plant life (e.g. low availability of the essential nutrients N, P, K and Ca and high concentrations of potentially phytotoxic Mg and Ni). Ultramafic soil diversity linked to the age of the soil or the degree of serpentinisation would thus be a main factor of plant diversity and distribution. The diverse topography of Kinabalu Park (ultramafic soils present between 400 and 2950 m asl) has given rise to high pedodiversity with the broad overall ultramafic soil types being: (i) deep laterite soils (Geric Ferralsols); (ii) moderately deep montane soils (Dystric Cambisols) with mor humus; (iii) shallow skeletal soils at high altitude (Eutric Cambisols Hypermagnesic); and (iv) bare serpentinite soils (Hypereutric Leptosols Hypermagnesic) at low altitude (200–700 m asl). Leptosols on serpentinite and Eutric Cambisols have the most extreme chemical properties in the whole Kinabalu Park area both with very high Mg:Ca molar quotients, with either high available Ni (Cambisols) or high pH (Leptosols). These soils host specific and adapted vegetation (high level of endemism) that tolerates geochemical peculiarities, including Ni hyperaccumulators. Geric Ferralsols present far less chemical constraints than Hypermagnesian Cambisols soils to the vegetation and host a tall and very diverse rainforest, not so different than that on non-ultramafic soils. It therefore appears that altitude, soil age and degree of bedrock serpentinisation are the main determining factors of soil properties: the qualifier “ultramafic” alone is not sufficient to define soil geochemical and ecological conditions in the Kinabalu Park area, probably more than in any other ultramafic region in the world

Predicting the degree of phosphorus saturation using the ammonium acetate-EDTA soil test

As a result of the important role played by phosphorus (P) in surface water eutrophication, the susceptibility of soils to release P requires evaluation. The degree of phosphorus saturation, assessed by oxalate extraction (DPS(ox)), has been used as an indicator. However, most laboratories do not include DPS(ox) in routine soil tests because of cost and time. This study evaluates the suitability of the ammonium acetate extraction in the presence of EDTA (AAEDTA), the standard soil test P (STP) in Wallonia (Southern Belgium), to predict DPS(ox); we also compared it with the Mehlich 3 extraction. Ninety-three topsoil samples were collected in agricultural soils throughout Wallonia. Good correlations were found between the AAEDTA and the Mehlich 3 methods for P, Fe and Al (r = 0.85, 0.77 and 0.86, respectively). An exponential relationship was found between P(AAEDTA) and DPS(ox). Results of principal component analysis and regression demonstrated that STP can be used to predict DPS(ox) (r = 0.93) after logarithmic transformation. Soil test Al was also a good indicator of the P sorption capacity (PSC(ox)) of soils (r = 0.86). Including the clay fraction in regression equations only slightly improved the prediction of PSC(ox) (r = 0.90), while other readily available data (such as pH or organic carbon) did not significantly improve either DPS(ox) or PSC(ox) predictions