Sreejith Raran-Kurussi

Semi-synthetic Pf RALP1-C1, the C-terminal region of the indispensable Pf RALP1 protein, competitively blocks merozoite invasion of RBCs. This hard-to-express malaria antigen was produced via 3-segment native chemical ligation.

Rapid developments in magic-angle-spinning (MAS) hardware over the past two decades have made possible the acquisition of high-resolution spectra of protons in solids, fuelling studies of small and large molecules alike. Nevertheless, proton resolution, limited by the strong dipole–dipole coupling network, remains a bottleneck even at MAS frequencies exceeding 100 kHz. We present here techniques based on phase-modulated homonuclear decoupling that dramatically improve proton coherence times and resolution compared to those of 60–95 kHz MAS alone using low average radiofrequency amplitudes (<100 kHz). A relatively high sensitivity (40–70%) and a straightforward optimization procedure performed directly on the sample being studied allow these gains to be realized in large biomolecules, as demonstrated here on a 326-residue cytoskeletal protein in its filamentous state. These techniques enable experiments with improved resolution on biomolecules while simultaneously taking advantage of the higher sensitivity available on probes with relatively large rotor volumes that cannot reach higher MAS frequencies.

High-resolution ¹H NMR of rigid solids is now routinely observed under fast magic angle sample spinning (MAS). Nevertheless, the spectral resolution of ¹H nuclei bonded to ¹⁴N is often compromised by residual dipolar splitting (RDS) and scalar coupling between ¹H and ¹⁴N. Given the ubiquity of the NH moiety and the high natural abundance of both nuclei, RDS broadening poses a widespread practical challenge. Heteronuclear ¹⁴N decoupling during ¹H acquisition is therefore essential for enhancing the spectral resolution. Previously, we demonstrated that low-power ¹⁴N CW decoupling via 70 kHz MAS improves the ¹H signal intensity by ∼20% and narrows ¹H line widths by ∼180 Hz but under stringent on-resonance ¹⁴N irradiation. Offset-tolerant ¹⁴N decoupling sequences are necessary to achieve good decoupling in samples with multiple ¹⁴N sites. Here, we assess several amplitude-modulated decoupling schemes, adapted from solution NMR. Experiments and numerical simulations determine that SUSAN1 (with t_p = 10 μs and ν_¹⁴N = 15-23 kHz) provides the best broadband ¹⁴N decoupling performance. Furthermore, an empirical correlation is established between pulse length and selective band-limited and non-selective broadband ¹⁴N decoupling behavior under low-power and fast MAS conditions.

The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a significant risk to global health, warranting the formulation of efficient preventive and therapeutic measures to tackle its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of coronaviruses plays a pivotal role in viral attachment and entry into host cells. The receptor-binding domain (RBD) of the SARS-CoV-2 S protein has demonstrated a robust binding affinity to ACE2 receptors in humans. Consequently, it has become a prime target for therapeutic interventions using antibodies, vaccines, or other designed inhibitors. This paper presents an optimized RBD sequence that can be efficiently expressed in Escherichia coli and refolded to yield a functional protein. Using optimized refolding procedures, we obtained 10-12 mg of active protein from a one-liter LB culture. The biological activity of the refolded RBD was confirmed by monitoring its interaction with the designed LCB1 miniprotein ligand by surface plasmon resonance, wherein they exhibited significant affinity levels as reflected by their dissociation constants (K _D s < 10 nM). The resulting RBD could be an ideal target for designing potent COVID-19 antivirals.

A cost-effective and robust diagnosis of an infectious agent is essential during a pandemic outbreak. While RT-qPCR-based methods are widely used, alternative methods that can be used outside the laboratory setup would enable better disease control and widen the surveillance efforts. The loop-mediated isothermal amplification (LAMP) method is an alternate method that has not yet been commercially deployed widely. The colorimetric LAMP can be adapted for various pathogens and would be helpful for high-throughput screening in a limited resource setting. Moreover, it is quicker and less labor-intensive. In our study, improved RT-LAMP assay primer sets were designed to detect SARS-CoV-2: N gene and ORF1a gene fragments. RT-LAMP assay primers were also designed to detect human RNase P to ensure RNA integrity. RT-LAMP reaction tubes/plates were incubated at 65°C for 30 min. Cell phone cameras were used to record results. RT-LAMP results were assessed by comparing the results obtained with RT-qPCR. RT-LAMP detected ~100 RNA copies, and its sensitivity was on par with RT-qPCR. Out of 60 patient samples with extracted RNA, a positive agreement of 98.2% was observed between RT-LAMP and RT-qPCR. On a cautionary note, severe discrepancies were observed if the same oligos were procured from different vendors, drastically affecting the test results. Therefore, mandatory quality control of primers is indispensable.

Proteins are dynamic molecules, relying on conformational changes to carry out function. Measurement of these conformational changes can provide insight into how function is achieved. For proteins in the solid state, this can be done by measuring the decrease in the strength of anisotropic interactions due to motion-induced fluctuations. The measurement of one-bond heteronuclear dipole-dipole coupling at magic-angle-spinning (MAS) frequencies >60 kHz is ideal for this purpose. However, rotational-echo double resonance (REDOR), an otherwise gold-standard technique for the quantitative measurement of these couplings, is difficult to implement under these conditions, especially in nondeuterated samples. We present here a combination of strategies based on REDOR variants ϵ-REDOR and DEDOR (deferred REDOR) and simultaneously measure residue-specific ¹⁵N-¹H and ¹³C_α-¹H_α dipole-dipole couplings in nondeuterated systems at the MAS frequency of 100 kHz. These strategies open up avenues to access dipolar order parameters in a variety of systems at the increasingly fast MAS frequencies that are now available.