Rigidity analysis of protein structures and rapid simulations of protein motion

It is a common goal in biophysics to understand protein structural properties and their relationship to protein function. I investigated protein structural properties using three coarse graining methods: a rigidity analysis method First, a geometric simulation method Froda and normal mode analysis as implemented in Elnemo to identify the protein directions of motion. Furthermore, I also compared the results between the coarse graining methods with the results from molecular dynamics and from experiments that I carried out. The results from the rigidity analysis across a set of protein families presented in chapter 3 highlighted two different patterns of protein rigidity loss, i.e. "sudden" and "gradual". It was found that theses characteristic patterns were in line with the rigidity distribution of glassy networks. The simulations of protein motion by merging flexibility, rigidity and normal mode analyses presented in chapter 4 were able to identify large conformational changes of proteins using minimal computational resources. I investigated the use of RMSD as a measure to characterise protein motion and showed that, despite it is a good measure to identify structural differences when comparing the same protein, the use of extensive RMSD better captures the extend of motion of a protein structure. The in-depth investigation of yeast PDI mobility presented in chapter 5 confirmed former experimental results that predicted a large conformational change for this enzyme. Furthermore, the results predicted: a characteristic rigidity distribution for yeast PDI, a minimum and a maximum active site distance and a relationship between the energy cutoff, i.e. the number of hydrogen bonds part of the network of bonds, and protein mobility. The results obtained were tested against molecular dynamics simulations in chapter 6. The MD simulation also showed a large conformational change for yeast PDI but with a slightly different minimum and maximum inter-cysteine distance. Furthermore, MD was able to reveal new data, i.e. the most likely inter-cysteine distance. In order to test the accuracy of the coarse graining and MD simulations I carried out cross-linking experiments to test the minimum inter-cysteine distance predictions. The results presented in chapter 7 show that human PDI minimum distance is below 12Å whereas the yeast PDI minimum distance must be above 12Å as no cross-linking structures where found with the available (12Å long) cross-linkers

The flexibility and dynamics of protein disulphide-isomerase

We have studied the mobility of the multi-domain folding catalyst, protein disulphide-isomerase (PDI), by a coarse-graining approach based on exibility. We analyse our simulations of yeast PDI (yPDI) using measures of backbone movement, relative positions and orientations of domains, and distances between functional sites. We nd that there is interdomain exibility at every interdomain junction but these show very di erent characteristics. The extent of interdomain exibility is such that yPDI's two active sites can approach much more closely than is found in crystal structures | and indeed hinge motion to bring these sites into proximity is the lowest energy normal mode of motion of the protein. The exibility predicted for yPDI (based on one structure) includes the other known conformation of yPDI and is consistent with (i) the mobility observed experimentally for mammalian PDI and (ii) molecular dynamics. We also observe intradomain exibility and clear di erences between the domains in their propensity for internal motion. Our results suggest that PDI exibility enables it to interact with many di erent partner molecules of widely di erent sizes and shapes, and highlights considerable similarities of yPDI and mammalian PDI

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Rigidity analysis of HIV-1 protease

We present a rigidity analysis on a large number of X-ray crystal structures of the enzyme HIV-1 protease using the 'pebble game' algorithm of the software first. We find that although the rigidity profile remains similar across a comprehensive set of high resolution structures, the profile changes significantly in the presence of an inhibitor. Our study shows that the action of the inhibitors is to restrict the exibility of the beta-hairpin flaps which allow access to the active site. The results are discussed in the context of full molecular dynamics simulations as well as data from NMR experiments

Global attitudes in the management of acute appendicitis during COVID-19 pandemic: ACIE Appy Study

Background: Surgical strategies are being adapted to face the COVID-19 pandemic. Recommendations on the management of acute appendicitis have been based on expert opinion, but very little evidence is available. This study addressed that dearth with a snapshot of worldwide approaches to appendicitis. Methods: The Association of Italian Surgeons in Europe designed an online survey to assess the current attitude of surgeons globally regarding the management of patients with acute appendicitis during the pandemic. Questions were divided into baseline information, hospital organization and screening, personal protective equipment, management and surgical approach, and patient presentation before versus during the pandemic. Results: Of 744 answers, 709 (from 66 countries) were complete and were included in the analysis. Most hospitals were treating both patients with and those without COVID. There was variation in screening indications and modality used, with chest X-ray plus molecular testing (PCR) being the commonest (19\ub78 per cent). Conservative management of complicated and uncomplicated appendicitis was used by 6\ub76 and 2\ub74 per cent respectively before, but 23\ub77 and 5\ub73 per cent, during the pandemic (both P < 0\ub7001). One-third changed their approach from laparoscopic to open surgery owing to the popular (but evidence-lacking) advice from expert groups during the initial phase of the pandemic. No agreement on how to filter surgical smoke plume during laparoscopy was identified. There was an overall reduction in the number of patients admitted with appendicitis and one-third felt that patients who did present had more severe appendicitis than they usually observe. Conclusion: Conservative management of mild appendicitis has been possible during the pandemic. The fact that some surgeons switched to open appendicectomy may reflect the poor guidelines that emanated in the early phase of SARS-CoV-2