Optimization of TROSY- and anti-TROSY-based 15N CPMG relaxation dispersion experiments through phase cycling.

CPMG relaxation dispersion studies of biomolecular dynamics on the µs-ms timescale can provide detailed kinetic, thermodynamic, and structural insights into function. Frequently, the 15N spin serves as the probe of choice, as uniform incorporation of the 15N isotope is facile and cost-effective, and the interpretation of the resulting data is often relatively straightforward. In conventional CPMG relaxation dispersion experiments the application of CPMG pulses with constant radiofrequency (RF) phase can lead to artifactual dispersion profiles that result from off-resonance effects, RF field inhomogeneity, and pulse miscalibration. The development of CPMG experiments with the [0013]-phase cycle has significantly reduced the impact of pulse imperfections over a greater bandwidth of frequency offsets in comparison to constant phase experiments. Application of 15N-TROSY-based CPMG schemes to studies of the dynamics of large molecules is necessary for high sensitivity, yet the correct incorporation of the [0013]-phase cycle is non-trivial. Here we present TROSY- and anti-TROSY-based 15N CPMG experiments with the [0013]-phase cycling scheme and demonstrate, through comprehensive numerical simulations and experimental validation, enhanced resistance to pulse imperfections relative to traditional schemes utilizing constant phase CPMG pulses. Notably, exchange parameters derived from the new experiments are in good agreement with those obtained using other, more established, 15N-based CPMG approaches.

Backbone resonance assignments and dynamics of S. cerevisiae SERF.

Abnormal protein aggregation and precipitation are associated with the perturbation of cellular function and underlie a variety of neurodegenerative diseases. S. cerevisiae SERF (ScSERF), a homolog of modifier of aggregation-4 (MOAG-4) and small EDRK-rich factor protein (SERF1a) is highly conserved and discovered as an enhancer of amyloid formation of Aß40 and α-synuclein both in vitro and in vivo. However, the detailed molecular mechanism whereby ScSERF and its homologs accelerate amyloid formation is not well known yet. Herein, we present the 1 H, 15 N and 13 C NMR assignments of the 68 amino acids long ScSERF. Although ScSERF displays a very high degree of disorder, secondary chemical shifts of Cα, Cß, 15 N{1 H}-NOE values and the residue-specific secondary structure propensity (SSP) scores indicate the segment spanning residues 36E-65 K has a strong helical propensity. This work sets the stage for further detailed structural and dynamic investigations of ScSERF and the molecular mechanism it utilizes in accelerating amyloid formation.

Molecular Insight into the Extracellular Chaperone Serum Albumin in Modifying the Folding Free Energy Landscape of Client Proteins.

Serum albumin (SA) is the most abundant extracellular chaperone protein presenting in various bodily fluids. Recently, several studies have revealed molecular mechanisms of SA in preventing the amyloid formation of amyloidogenic proteins. However, our insight into the mechanism SA employed to sense and regulate the folding states of full-length native proteins is still limited. Addressing this question is technically challenging due to the intrinsic dynamic nature of both chaperones and clients. Here using nuclear magnetic resonance spectroscopy, we show SA modifies the folding free energy landscape of clients and subsequently alters the equilibria between different compact conformations of its clients, resulting in the increased populations of excited states of client proteins. This modulation of client protein conformation by SA can change the client protein activity in a way that cannot be interpreted on the basis of its ground state structure; therefore, our work provides an alternative insight of SA in retaining a balanced functional proteome.

Improved performance in γ-polyglutamic acid production by Bacillus subtilis LX on industrial scale by impeller retrofitting and its unstructured microbial growth kinetics model.

We conducted industrial scale γ-polyglutamic acid (γ-PGA) production by Bacillus subtilis (B. subtilis) LX and modeled its microbial growth kinetics based on a logistic regression. We found that the use of a three-layer impeller including a lower semicircular disc impeller and two-layers of six-wide-leaf impellers were able to both increase γ-PGA yields and decrease fermentation time as compared with two-layer Rushton impellers. Indeed, our results revealed that the optimal γ-PGA yield (20.67 ± 2.19 g/L) was obtained after 40 hr in the impeller retrofitted fermenter, and this yield was 29.7% higher than that in Rushton impellers fixed fermenter. The microbial growth kinetics of B. subtilis LX in this system were established, and the model was consistent with the experimental data (R2 = 0.924) suggesting that it was suitable for describing the microbial growth kinetics underlying γ-PGA production on an industrial scale. In addition, biomass yield (Yx/s-glucose), γ-PGA yield (Yp/s-glucose), γ-PGA yield (Yp/s-glutamate), and the correlation between γ-PGA production and B. subtilis LX (Yp/x) were found to be 0.043, 0.133, 0.743, and 3.090 g/g, respectively, in the impeller retrofitted fermenter, as compared with 0.036, 0.103, 0.629, and 2.819 g/g, respectively, in the two-layer Rushton impeller fermenter.