Repressor of primer (Rop, or ROM, RNA I modulator) is a 63 amino acid four-helix bundle protein that exists in solution as an anti-parallel homodimer. This protein has been extensively studied, ...including by X-ray crystallography, NMR, rational design, and combinatorial mutagenesis. Previous NMR experiments with wild-type Rop were carried out at pH 2.3 and pH 6.3. In this paper, we report complete N–H backbone assignments for three variants of Rop under the same pH 6.3 conditions: wild-type Rop; a cysteine-free pseudo-wild type variant (C38A C52V); and a core-repacked variant of the Cys-free variant (T19V L41V C38A C52V). These assignments enable functional and dynamic studies of wild-type and Cys-free variants of Rop.
Abstract
Malaria is a vector-borne disease caused by parasites of the Plasmodium genus that infect millions of people annually, with P. falciparum being the most common and deadliest species. ...Development of immunological protection against clinical malaria requires chronic or repetitive parasite exposure, is mediated by IgG, and is correlated with antibodies against proteins expressed by merozoites, the free form of the parasite that invades erythrocytes. The goal of our study was to better understand the development of this protective antibody response, using merozoite surface protein 1 (MSP1) as a model antigen. MSP1 is highly abundant on the merozoite surface, highly immunogenic, and highly variable between parasite strains. Using B cell tetramers, we isolated MSP1-specific IgM+ and IgG+ memory B cells (MBCs) from immune adults and non-immune children living in Tororo, Uganda, a region of high transmission. In children, MSP1-specific B cells were mainly unswitched (IgM+) classical MBCs (cMBCs; CD21+ CD27+), while adults showed enrichment for class-switched (IgG+) cMBCs. Sequence analysis of the heavy chain variable regions revealed that MSP1-specific MBCs from adults carried higher rates of somatic hypermutation and showed extensive clonal expansion compared to those from children. When expressed as IgG, antibodies from IgM+ MBCs showed low avidity to MSP1, while those from IgG+ MBCs displayed strong reactivity with recombinant MSP1 and whole merozoites and inhibited parasite growth. These results suggest that extensive evolution of B cell responses takes place as a result of life-long P. falciparum exposure, which may be essential for the development of protection against malaria.
Both the prediction and design of protein structure, using computational and rational approaches, remain significant challenges in protein chemistry. A major limitation to developing a comprehensive ...physicochemical model of the protein structure‐sequence relationship is the vastness of sequence space and the low‐throughput nature of biophysical studies. We are pursuing a combinatorial approach to understand better the sequence structure‐relationship: sorting large libraries of protein variants for structured proteins. We have developed a high‐throughput cell‐based screen for activity of the well‐studied four‐helix bundle protein Rop. To collect quantitative stability data for large numbers of variants, we have developed a method of high‐throughput hydrophobic dye binding called High‐Throughput Thermal Scanning (HTTS) which can be applied using automation and a real‐time PCR machine 96‐wells at a time. This system is being used to directly test the “rules” of protein design, taking those rules as hypotheses and sorting the resulting libraries for structure and stability. Here we will discuss the in‐depth analysis of a library of hydrophobic core variants, as well as initial results from surface libraries and rational design based on library statistics. This work was supported by a grant from the NIH (R01 GM083114) to TJM.
The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the urgent need for assays that detect protective levels of neutralizing antibodies. We studied the ...relationship among anti-spike ectodomain (anti-ECD), anti-receptor-binding domain (anti-RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by 2 in vitro assays using convalescent plasma samples from 68 patients with COVID-19. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers and in vitro VN titers. The probability of a VN titer of ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment, was ≥80% when anti-RBD or anti-ECD titers were ≥1:1350. Of all donors, 37% lacked VN titers of ≥160. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease VN or IgG titers. Analysis of 2814 asymptomatic adults found 73 individuals with anti-ECD IgG titers of ≥1:50 and strong positive correlation with anti-RBD and VN titers. Fourteen of these individuals had VN titers of ≥1:160, and all of them had anti-RBD titers of ≥1:1350. We conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titers of ≥1:1350 may provide critical information about protection against COVID-19 disease.
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used ...with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.
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