Electrostatic effects in proteins are governed by redistribution of the conformational ensemble

The ability to regulate pH dynamics is one of the hallmarks of all living cells. pH affects biological function predominantly through its effect on proteins, through the binding or release of H+ to ionizable groups. To understand the structural basis of the pH sensitivity of proteins, it is necessary to understand the molecular basis of H+ binding events. The H+-binding affinities of ionizable groups (i.e. pKa values) are known to be sensitive to the properties of their microenvironments. The inability of sophisticated computational methods for structure-based calculations of pKa values to reproduce the experimentally measured pKa values suggest that H+ binding to proteins is a more complex process than currently acknowledged. To examine in detail the molecular determinants of pKa values, I studied variants of staphylococcal nuclease with buried Lys residues. These buried Lys residues were chosen because they have highly anomalous pKa values (depressed, which favors the neutral state) that are difficult to reproduce with structure-based pKa calculations. In dehydrated environments, electrostatic effects such as Coulomb interactions and dehydration energies are stronger relative to those on the well hydrated protein surface and are highly sensitive to the polarity and polarizability of their environments. The studies described in this dissertation examined the hypothesis that the magnitude of electrostatic effects in proteins are not as severely influenced by the properties of the microenvironments of ionizable moieties as they are by the propensity of the conformational ensemble to redistribute in response to changes in pH. In chapter 2, the physical properties of the buried Lys residues were probed using 15N NMR spectroscopy. In most cases, pH-dependent structural reorganization was evident in the pH titrations tracked with the 15Nzeta resonance of the buried Lys residues. In chapter 3, the nature of this reorganization was characterized in 25 variants of SNase with internal Lys residues. These studies used backbone-based NMR spectroscopy experiments. The results of this work demonstrate that the apparent pKa values of the Lys residues measured by linkage thermodynamics report on pH-driven redistribution of the conformational ensemble. The redistribution favors conformational states in which the charged Lys side chains are exposed to solvent. By correlating thermodynamic data (i.e. stability) with detailed structural information about the partially unfolded states that SNase can access, it was possible to map the conformational ensemble of this protein with unprecedented resolution. In chapter 4, the structural and thermodynamic consequences of buried ion pairs in SNase was examined. NMR spectroscopy and X-ray crystallography showed various degrees of structural reorganization that lead to the hydration of at least one of the buried groups. These results demonstrate that not only does structural reorganization govern the properties of individual buried groups, structural reorganization can also determine the properties of buried ion pairs. In chapter 5 I provide irrefutable evidence of strong apparent interactions between buried and surface ionizable groups. NMR spectroscopy and equilibrium thermodynamic experiments demonstrated that the interactions between internal and surface ionizable groups are not Coulomb interactions. Instead, the apparent interactions are mediated by conformational reorganization driven by the ionization of the buried residues. Most of what is known about protein electrostatics reflects insights from decades of structure-based calculations with continuum electrostatic models, and more recently with constant pH molecular dynamics calculations. The data presented in this dissertation demonstrate that conformational reorganization can be the dominant factor that determines the pKa values of ionizable groups in proteins. Structure-based prediction of conformational reorganization remains extremely challenging; my results suggest an explanation for the short-comings of structure-based calculations to reproduce experimental pKa values and other electrostatic properties of proteins.

Electrostatic effects in proteins are governed by redistribution of the conformational ensemble

The ability to regulate pH dynamics is one of the hallmarks of all living cells. pH affects biological function predominantly through its effect on proteins, through the binding or release of H+ to ionizable groups. To understand the structural basis of the pH sensitivity of proteins, it is necessary to understand the molecular basis of H+ binding events. The H+-binding affinities of ionizable groups (i.e. pKa values) are known to be sensitive to the properties of their microenvironments. The inability of sophisticated computational methods for structure-based calculations of pKa values to reproduce the experimentally measured pKa values suggest that H+ binding to proteins is a more complex process than currently acknowledged. To examine in detail the molecular determinants of pKa values, I studied variants of staphylococcal nuclease with buried Lys residues. These buried Lys residues were chosen because they have highly anomalous pKa values (depressed, which favors the neutral state) that are difficult to reproduce with structure-based pKa calculations. In dehydrated environments, electrostatic effects such as Coulomb interactions and dehydration energies are stronger relative to those on the well hydrated protein surface and are highly sensitive to the polarity and polarizability of their environments. The studies described in this dissertation examined the hypothesis that the magnitude of electrostatic effects in proteins are not as severely influenced by the properties of the microenvironments of ionizable moieties as they are by the propensity of the conformational ensemble to redistribute in response to changes in pH. In chapter 2, the physical properties of the buried Lys residues were probed using 15N NMR spectroscopy. In most cases, pH-dependent structural reorganization was evident in the pH titrations tracked with the 15Nzeta resonance of the buried Lys residues. In chapter 3, the nature of this reorganization was characterized in 25 variants of SNase with internal Lys residues. These studies used backbone-based NMR spectroscopy experiments. The results of this work demonstrate that the apparent pKa values of the Lys residues measured by linkage thermodynamics report on pH-driven redistribution of the conformational ensemble. The redistribution favors conformational states in which the charged Lys side chains are exposed to solvent. By correlating thermodynamic data (i.e. stability) with detailed structural information about the partially unfolded states that SNase can access, it was possible to map the conformational ensemble of this protein with unprecedented resolution. In chapter 4, the structural and thermodynamic consequences of buried ion pairs in SNase was examined. NMR spectroscopy and X-ray crystallography showed various degrees of structural reorganization that lead to the hydration of at least one of the buried groups. These results demonstrate that not only does structural reorganization govern the properties of individual buried groups, structural reorganization can also determine the properties of buried ion pairs. In chapter 5 I provide irrefutable evidence of strong apparent interactions between buried and surface ionizable groups. NMR spectroscopy and equilibrium thermodynamic experiments demonstrated that the interactions between internal and surface ionizable groups are not Coulomb interactions. Instead, the apparent interactions are mediated by conformational reorganization driven by the ionization of the buried residues. Most of what is known about protein electrostatics reflects insights from decades of structure-based calculations with continuum electrostatic models, and more recently with constant pH molecular dynamics calculations. The data presented in this dissertation demonstrate that conformational reorganization can be the dominant factor that determines the pKa values of ionizable groups in proteins. Structure-based prediction of conformational reorganization remains extremely challenging; my results suggest an explanation for the short-comings of structure-based calculations to reproduce experimental pKa values and other electrostatic properties of proteins.

The surgical safety checklist and patient outcomes after surgery: a prospective observational cohort study, systematic review and meta-analysis

© 2017 British Journal of Anaesthesia Background: The surgical safety checklist is widely used to improve the quality of perioperative care. However, clinicians continue to debate the clinical effectiveness of this tool. Methods: Prospective analysis of data from the International Surgical Outcomes Study (ISOS), an international observational study of elective in-patient surgery, accompanied by a systematic review and meta-analysis of published literature. The exposure was surgical safety checklist use. The primary outcome was in-hospital mortality and the secondary outcome was postoperative complications. In the ISOS cohort, a multivariable multi-level generalized linear model was used to test associations. To further contextualise these findings, we included the results from the ISOS cohort in a meta-analysis. Results are reported as odds ratios (OR) with 95% confidence intervals. Results: We included 44 814 patients from 497 hospitals in 27 countries in the ISOS analysis. There were 40 245 (89.8%) patients exposed to the checklist, whilst 7508 (16.8%) sustained ≥1 postoperative complications and 207 (0.5%) died before hospital discharge. Checklist exposure was associated with reduced mortality [odds ratio (OR) 0.49 (0.32–0.77); P\u3c0.01], but no difference in complication rates [OR 1.02 (0.88–1.19); P=0.75]. In a systematic review, we screened 3732 records and identified 11 eligible studies of 453 292 patients including the ISOS cohort. Checklist exposure was associated with both reduced postoperative mortality [OR 0.75 (0.62–0.92); P\u3c0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61–0.88); P\u3c0.01; I2=89%). Conclusions: Patients exposed to a surgical safety checklist experience better postoperative outcomes, but this could simply reflect wider quality of care in hospitals where checklist use is routine

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally

Critical care admission following elective surgery was not associated with survival benefit: prospective analysis of data from 27 countries

This was an investigator initiated study funded by Nestle Health Sciences through an unrestricted research grant, and by a National Institute for Health Research (UK) Professorship held by RP. The study was sponsored by Queen Mary University of London