Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts

Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism

Differences in bacterial diversity and abundance between nest components.

<p>Differences between the four nest components in 1) diversity of OTUs per sample using ARISA and 2) abundance of bacteria per sample quantified using culturing techniques. Data are presented as predicted means ± standard error. Cultured bacteria abundance means were calculated using log₁₀ transformed data. Proportions of haemolytic bacteria are stated as percentage. “Preincubation” and “Incubated” indicate eggs sampled before and during the incubation period, respectively. Generalised linear mixed models were used due to the non-independence of samples collected from the same nest.</p><p>Differences in bacterial diversity and abundance between nest components.</p

Differences in bacterial assemblages between the nest components.

<p>Non-metric multi-dimensional scaling along A) the first and second axis, B) the second and third axis and C) the first and third axis, displaying differences in bacterial assemblages between the four nest components including preincubation eggs (closed circles and dash dot line), incubated eggs (open circles and long dashed line), nestling faeces (closed triangles and dotted line) and nesting material (open diamonds and a solid line).</p

Prevalence of cultured bacteria in different nest components.

<p>Prevalence of cultured bacteria between nest components, including <i>Enterococcus</i> spp. (dark grey), coliforms (white), <i>Staphylococcus/Streptococcus</i> spp. (black) and <i>Escherichia coli/Staphylococcus saprophyticus</i> (light grey).</p

Mean Bray-Curtis similarities between nest components.

<p>Data are presented as means ± standard error. “Preincubation” and “Incubated” indicate eggs sampled before and during the incubation period, respectively.</p><p>Mean Bray-Curtis similarities between nest components.</p

Abundance of cultured bacteria in preincubation and incubated eggs.

<p>Abundance of cultured bacteria sampled from eggs before and during incubation, including haemolytic bacteria (Ha), non-haemolytic bacteria (nHa), <i>Enterococcus</i> (En), Coliforms (Co), <i>Streptococcus</i>/<i>Staphylococcus</i> (SS) and <i>Escherichia coli/Staphylococcus saprophyticus</i> (Es/Ss). Bars represent predicted means ± SE from GLMMs, including sampling date as a covariate.</p