GADIS: Algorithm for designing sequences to achieve target secondary structure profiles of intrinsically disordered proteins.

Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding to the binding interface; and (iii) maintaining the native amino acid composition. Here, we report the development of a G: enetic A: lgorithm for D: esign of I: ntrinsic secondary S: tructure (GADIS) for designing sequences that satisfy the specified constraints. We describe the algorithm and present results to demonstrate the applicability of GADIS by designing sequence variants of the intrinsically disordered PUMA system that undergoes coupled folding and binding to Mcl-1. Our sequence designs span a range of intrinsic helicity profiles. The predicted variations in sequence-encoded mean helicities are tested against experimental measurements.The US-National Science Foundation and US-National Institutes of Health supported this work through grants MCB-1121867 and 5RO1 NS056114, respectively to R.V.P. J.C. and S.L.S. were supported by the Wellcome Trust (WT 095195MA). M.D.C. was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship.This is the author accepted manuscript. The final version is available from Oxford University Press via http://dx.doi.org/10.1093/protein/gzw03

Home-based system for physical activity monitoring in patients with multiple sclerosis (Pilot study)

Background Limitations in physical activity are considered as a key problem in patients with multiple sclerosis (PwMS). Contemporary methods to assess the level of physical activity in PwMS are regular clinical observation. However, these methods either rely on high recall and accurate reporting from the patients (e.g. self-report questionnaires), or they are conducted during a particular clinical assessment with predefined activities. Therefore, the main aim of this pilot study was to develop an objective method to gather information about the real type and intensity of daily activities performed by PwMS in every-day living situations using an accelerometer. Furthermore, the accelerometer-derived measures are investigated regarding their potential for discriminating between different MS groups. Methods Eleven PwMS that were able to walk independently (EDSS&#8201;&#8804;&#8201;5) were divided into two groups: mild disability (EDSS 1&#8211;2.5; n&#8201;=&#8201;6) and moderate disability (EDSS 3 &#8211;5; n&#8201;=&#8201;5). Participants made use of an activity monitor device attached to their waist during their normal daily activities over 4 measurements. Activity parameters were assessed and compared for the time of each participant&#8217;s first measurement and follow-up measurement. Furthermore, differences between both subgroups, and the correlation of activity parameters with the clinical neurological variable (EDSS) were investigated. Results Participants showed significant decline in step count (p&#8201;=&#8201;0.008), maximum walking speed (p&#8201;=&#8201;0.02) and physical activity intensity (p&#8201;=&#8201;0.03) throughout the study period. Compared to the mild subgroup, moderate affected participant accumulated less number of steps (G1: 9214.33&#8201;±&#8201;2439.11, G2: 5018.13&#8201;±&#8201;2416.96; p&#8201;<&#8201;0.005) and were slower (G1: 1.48&#8201;±&#8201;0.19, G2: 1.12&#8201;±&#8201;0.44; p&#8201;=&#8201;0.03). Additionally, the EDSS correlated negatively with mean walking speed (r&#8201;=&#8201;- 0.71, p&#8201;=&#8201;0.01) and steps count (r&#8201;=&#8201;- 0.54, p&#8201;=&#8201;0.08). Conclusions In this study, we used a portable activity monitoring sensor to gather information about everyday physical activity in PwMS at home. We showed that objective measurements using simple 3D accelerometers can track daily physical activity fluctuation. Furthermore, they track disability changes better than clinical measures. Thus, they can help to develop activity based treatments for PwMS

Metastability of native proteins and the phenomenon of amyloid formation

An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable. © 2011 American Chemical Society

Intrinsic determinants of neurotoxic aggregate formation by the amyloid β peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ40 peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ42 peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide. © 2010 by the Biophysical Society

Increased RPA1 gene dosage affects genomic stability potentially contributing to 17p13.3 duplication syndrome

A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression

A Systematic Literature Review of Students as Partners in Higher Education

“Students as Partners” (SaP) in higher education re-envisions students and staff as active collaborators in teaching and learning. Understanding what research on partnership communicates across the literature is timely and relevant as more staff and students come to embrace SaP. Through a systematic literature review of empirical research, we explored the question: How are SaP practices in higher education presented in the academic literature? Trends across results provide insights into four themes: the importance of reciprocity in partnership; the need to make space in the literature for sharing the (equal) realities of partnership; a focus on partnership activities that are small scale, at the undergraduate level, extracurricular, and focused on teaching and learning enhancement; and the need to move toward inclusive, partnered learning communities in higher education. We highlight nine implications for future research and practice.</jats:p

Consequences of inducing intrinsic disorder in a high-affinity protein-protein interaction

The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (Kd ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost

Altered Intracellular Localization and Mobility of SBDS Protein upon Mutation in Shwachman-Diamond Syndrome

Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients