Rubredoxin Variant Folds without Iron

Pyroccocus furiosus rubredoxin (PFRD), like most studied hyperthermophilic proteins, does not undergo reversible folding. The irreversibility of folding is thought to involve PFRD’s iron-binding site. Here we report a PFRD variant (PFRD-XC4) whose iron binding site was redesigned to eliminate iron binding using a computational design algorithm. PFRD-XC4 folds without iron and exhibits reversible folding with a melting temperature of 82 °C, a thermodynamic stability of 3.2 kcal mol^(-1) at 1 °C, and NMR chemical shifts similar to that of the wild-type protein. This variant should provide a tractable model system for studying the thermodynamic origins of protein hyperthermostability

Developing a Tool for Selection for Medical School : A search for academic and non-academic parameters to predict future medical school performance

Worldwide places in medical school are scarce and medical education and training are expensive for providers and learners. Therefore, medical schools aim to offer the places available only to those applicants with the highest probability of successful medical training and subsequent career. To reach this goal, medical schools have developed several selection procedures, including interviews, admission tests and other measures of personal competencies. Uniquely in the Netherlands, selection was organised nationally based on a lottery that is weighted for academic attainment. However, both the lottery and the unproven selection procedures have been described as unfair to medical school applicants, as neither includes any truly objective criteria for predicting future performance. The Dutch situation in which access to medical school was granted by lottery and the possibility to select up to 50% of the students by a selection procedure provided a unique opportunity to form a control group of randomly admitted students to compare with those selected. We developed an evidence-based selection procedure addressing non-academic (i.e. motivation) as well as academic skills. The former evaluated motivation through the determination of the candidate’s active involvement in extracurricular activities, the latter by tests concerning the study skills of candidates in a medical school context. The main outcome was that the relative risk for dropping out of medical school was significantly lower in selected students than in controls admitted by lottery. Those selected obtained a higher mean grade than the lottery admitted students on their clerkships. Thereby selected students participated more often in extracurricular activities, which was also associated with higher clerkship grades

Characterization of the Mechanosensitive Channel of Large Conductance

Osmoregulation is an essential process in bacteria and higher organisms regulated by the mechanosensitive ion channels. The mechanosensitive channel of large conductance (MscL) is an integral membrane protein that responds to pressure in an effort to prevent cell lysis during osmotic shock. Conversion of MscL from a membrane bound form to a water soluble form was attempted by three methods: computational design, random mutagenesis and chemical modification. The water soluble form of MscL was achieved with cysteine modification method. The stability, pH dependence, and C-terminal helix of MscL were also investigated. The structure of the cab beta-class carbonic anhydrase (Cab) has been determined to 2.1 A resolution. Cab exists as a dimer with a fold similar to plant beta-class carbonic anhydrases. The active site zinc is coordinated by Cys32, His87, and Cys90, with the tetrahedral coordination completed by a water molecule. The difference between plant and cab beta-class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes molecule near the active site, suggesting a proton transfer pathway to the solvent. The structure of the nitrogenase iron protein in the all-ferrous [4Fe-4S]0 form has been determined to 2.2 A resolution. The structure demonstrates that major conformational changes are not necessary to accommodate cluster reduction to the [4Fe-4S]0 state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands reveals that the [4Fe-4S] cluster of the iron protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the capability of existing in three oxidation states. The role of surface salt bridges in protein stabilization has been investigated. The NMR structure of a rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges is presented here. The analysis shows that the surface sidechain to sidechain salt bridge between does not stabilize PFRD-XC4. The mainchain to sidechain salt bridge, however, stabilizes PFRD-XC4 by 1.5 kcal mol-1. The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.</p

Ab initio molecular-replacement phasing for symmetric helical membrane proteins

An ab initio molecular-replacement method for phasing X-ray diffraction data for symmetric helical membrane proteins has been developed. The described method is based on generating all possible orientations of idealized transmembrane helices and using each model in a molecular-replacement search

Analytic models for mechanotransduction: gating a mechanosensitive channel

Analytic estimates for the forces and free energy generated by bilayer deformation reveal a compelling and intuitive model for MscL channel gating analogous to the nucleation of a second phase. We argue that the competition between hydrophobic mismatch and tension results in a surprisingly rich story which can provide both a quantitative comparison to measurements of opening tension for MscL when reconstituted in bilayers of different thickness and qualitative insights into the function of the MscL channel and other transmembrane proteins

The relationship between extracurricular activities assessed during selection and during medical school and performance

Several medical schools include candidates’ extracurricular activities in their selection procedure, with promising results regarding their predictive value for achievement during the clinical years of medical school. This study aims to reveal whether the better achievement in clinical training of students selected on the basis of their extracurricular activities could be explained by persistent participation in extracurricular activities during medical school (msECAs). Lottery-admitted and selected student admission groups were compared on their participation in three types of msECAs: (1) research master, (2) important board positions or (3) additional degree programme. Logistic regression was used to measure the effect of admission group on participation in any msECA, adjusted for pre-university GPA. Two-way ANCOVA was used to examine the inter-relationships between admission group, participation in msECAs and clerkship grade, with pre-university GPA as covariate. Significantly more selected students compared to lottery-admitted students participated in any msECA. Participation in msECAs was associated with a higher pre-university GPA for lottery-admitted students only, whereas participation in msECAs was associated with higher clerkship grades for selected students only. These results suggest that persistent participation in extracurricular activities of selected students favours better clinical achievement, supporting the inclusion of ECAs in the selection procedure. More insight in the rationale behind participation in extracurricular activities during medical school may explain differences found between lottery-admitted and selected students

An Autonomous CDR3δ is Sufficient for γδ T Cell Recognition of the Nonclassical MHC-I T10/T22

It remains unclear whether γδ T cell receptors (TCRs) detect antigens in a manner similar to antibodies or αβ TCRs. Here we show that reactivity between G8 and KN6 γδ TCRs and the MHC class Ib molecule T22 can be transplanted, with retention of wild-type ligand affinity, after en bloc grafting of G8 and KN6 CDR3δ loops in place onto the CDR3α loop of an αβ TCR. We also find that a shared sequence motif within CDR3δ loops of all T22-reactive γδ TCRs binds T22 in energetically distinct fashions, and that T10d, which binds G8 with weak affinity, is converted into a high-affinity ligand by a single point mutation. These results demonstrate an unprecedented autonomy of a single CDR3 loop in antigen recognition

Cosmologies of Selection:Lessons from the faculty biscuit tin

We conducted an ethnography of the faculty biscuit tin as we were interested in the lived experience of the biscuits contained within it. We used a constructivist epistemology, a social constructionist interpretive framework and a phenomenological methodology that included analysis from the perspectives of deixis and cosmology. The biscuits perceived that they were important to a selecting force and that the characteristics of one particular group had a specific value to the selector. Some enduring benefits may derive from the selection of this group, although its attractions were less immediately obvious than those of others. What is immediately attractive may not be the most fit for purpose; lessons for the selection of medical students may arise from this exploration of the selection experiences of biscuits in a faculty biscuit tin