Communication: Nanosecond folding dynamics of an alpha helix: Time-dependent 2D-IR cross peaks observed using polarization-sensitive dispersed pump-probe spectroscopy

We present a simple method to measure the dynamics of cross peaks in time-resolved two-dimensional vibrational spectroscopy. By combining suitably weighted dispersed pump-probe spectra, we eliminate the diagonal contribution to the 2D-IR response, so that the dispersed pump-probe signal contains the projection of only the cross peaks onto one of the axes of the 2D-IR spectrum. We apply the method to investigate the folding dynamics of an alpha-helical peptide in a temperature-jump experiment and find characteristic folding and unfolding time constants of 260 ± 30 and 580 ± 70 ns at 298 K

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems

Targeted resequencing of candidate genes using selector probes

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R2 = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation

In silico-designed lignin peroxidase from Phanerochaete chrysosporium shows enhanced acid stability for depolymerization of lignin

Background: The lignin peroxidase isozyme H8 from the white-rot fungus Phanerochaete chrysosporium (LiPH8) demonstrates a high redox potential and can efficiently catalyze the oxidation of veratryl alcohol, as well as the degradation of recalcitrant lignin. However, native LiPH8 is unstable under acidic pH conditions. This characteristic is a barrier to lignin depolymerization, as repolymerization of phenolic products occurs simultaneously at neutral pH. Because repolymerization of phenolics is repressed at acidic pH, a highly acid-stable LiPH8 could accelerate the selective depolymerization of recalcitrant lignin. Results: The engineered LiPH8 was in silico designed through the structural superimposition of surface-active site-harboring LiPH8 from Phanerochaete chrysosporium and acid-stable manganese peroxidase isozyme 6 (MnP6) from Ceriporiopsis subvermispora. Effective salt bridges were probed by molecular dynamics simulation and changes to Gibbs free energy following mutagenesis were predicted, suggesting promising variants with higher stability under extremely acidic conditions. The rationally designed variant, A55R/N156E-H239E, demonstrated a 12.5-fold increased half-life under extremely acidic conditions, 9.9-fold increased catalytic efficiency toward veratryl alcohol, and a 7.8-fold enhanced lignin model dimer conversion efficiency compared to those of native LiPH8. Furthermore, the two constructed salt bridges in the variant A55R/N156E-H239E were experimentally confirmed to be identical to the intentionally designed LiPH8 variant using X-ray crystallography (PDB ID: 6A6Q). Conclusion: Introduction of strong ionic salt bridges based on computational design resulted in a LiPH8 variant with markedly improved stability, as well as higher activity under acidic pH conditions. Thus, LiPH8, showing high acid stability, will be a crucial player in biomass valorization using selective depolymerization of lignin

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de-novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized