Combining experimental and simulation data to describe all of the structures and the pathways involved in folding a protein is problematical. Transition states can be mapped experimentally by phi values, but the denatured state is very difficult to analyse under conditions that favour folding. Also computer simulation at atomic resolution is currently limited to about a microsecond or less. Ultrafast-folding proteins fold and unfold on timescales accessible by both approaches, so here we study the folding pathway of the three-helix bundle protein Engrailed homeodomain. Experimentally, the protein collapses in a microsecond to give an intermediate with much native alpha-helical secondary structure, which is the major component of the denatured state under conditions that favour folding. A mutant protein shows this state to be compact and contain dynamic, native-like helices with unstructured side chains. In the transition state between this and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion-collision model. Molecular dynamics simulations give rate constants and structural details highly consistent with experiment, thereby completing the description of folding at atomic resolution.

⌽ values are used to map structures of protein-folding transition states from changes in free energies of denaturation (⌬⌬GD-N) and activation on mutation. A recent reappraisal proposed that ⌽ values for ⌬⌬GD-N < 1.7 kcal͞mol are artifactual. On discarding such derived ⌽ values from published studies, the authors concluded that there are no high ⌽ values in diffuse transition states, which are consequently uniformly diffuse with no evidence for nucleation. However, values of ⌬⌬GD-N > 1.7 kcal͞mol are often found for large side chains that make dispersed tertiary interactions, especially in hydrophobic cores that are in the process of being formed in the transition state. Conversely, specific local interactions that probe secondary structure tend to have ⌬⌬GD-N Ϸ 0.5-2 kcal͞mol. Discarding ⌽ values from lower-energy changes discards the crucial information about local interactions and makes transition states appear uniformly diffuse by overemphasizing the dispersed tertiary interactions. The evidence for the 1.7 kcal͞mol cutoff was based on mutations that had been deliberately designed to be unsuitable for ⌽-value analysis because they are structurally disruptive. We confirm that reliable ⌽ values can be derived from the recommended mutations in suitable proteins with 0.6 < ⌬⌬GD-N < 1.7 kcal͞mol, and there are many reliable high ⌽ values. Transition states vary from being rather diffuse to being well formed with islands of near-complete secondary structure. We also confirm that the structures of transition-state ensembles can be perturbed by mutations with ⌬⌬G D-N Ͼ Ͼ 2 kcal͞mol and that protein-folding transition states do move on the energy surface on mutation.barnase ͉ protein A ͉ nucleation-condensation ͉ framework ͉ Hammond T he ⌽-value analysis is a particular set of protein-engineering methods that is used to map the structures of transition states and intermediates in protein-folding, catalysis, binding, and conformational transitions of proteins at the level of individual residues (1-5). ⌽ is the ratio of change of free energy of activation for folding, ⌬⌬G ‡-D , to the equilibrium free energy of folding, ⌬⌬G N-D , † and scores the extent of formation of structure on a scale of 0 to 1 at the level of individual residues. ⌽ is similar but not identical to the constants ␣ or ␤ of classical rateequilibrium-free-energy relationships (REFERs) of covalentbond chemistry. Linear free-energy relationships are the classical means of analyzing the structures of transition states. The structure of a reagent is subtly altered by small changes, and the consequent perturbations of the kinetics and equilibrium of the reaction are measured. Under certain circumstances, which often rely on the chemist's judgement in making sensible structural changes, there can be a linear relationship between ⌬G ‡ , the change in activation energy, and ⌬G 0 , the change in equilibrium free energy; i.e., (Ѩ⌬G ‡ /Ѩstructure)͞(Ѩ⌬G 0 /Ѩstruc-ture) ϭ ␣ in an REFER (6) (or ϭ ␤ in the earlier Brønsted plots for catalysis). The ␣ (or ␤) value is...

We have assessed the published predictions of the pathway of folding of the B domain of protein A, the pathway most studied by computer simulation. We analyzed the transition state for folding of the three-helix bundle protein, by using experimental ⌽ values on some 70 suitable mutants. Surprisingly, the third helix, which has the most stable ␣-helical structure as a peptide fragment, is poorly formed in the transition state, especially at its C terminus. The protein folds around a nearly fully formed central helix, which is stabilized by extensive hydrophobic side chain interactions. The turn connecting the poorly structured first helix to the central helix is unstructured, but the turn connecting the central helix to the third is in the process of being formed as the N-terminal region of the third helix begins to coalesce. The transition state is inconsistent with a classical framework mechanism and is closer to nucleation-condensation. None of the published atomistic simulations are fully consistent with the experimental picture although many capture important features. There is a continuing need for combining simulation with experiment to describe folding pathways, and of continued testing to improve predictive methods.

Peri-operative SARS-CoV-2 infection increases postoperative mortality. The aim of this study was to determine the optimal duration of planned delay before surgery in patients who have had SARS-CoV-2 infection. This international, multicentre, prospective cohort study included patients undergoing elective or emergency surgery during October 2020. Surgical patients with pre-operative SARS-CoV-2 infection were compared with those without previous SARS-CoV-2 infection. The primary outcome measure was 30-day postoperative mortality. Logistic regression models were used to calculate adjusted 30-day mortality rates stratified by time from diagnosis of SARS-CoV-2 infection to surgery. Among 140,231 patients (116 countries), 3127 patients (2.2%) had a pre-operative SARS-CoV-2 diagnosis. Adjusted 30-day mortality in patients without SARS-CoV-2 infection was 1.5% (95%CI 1.4-1.5). In patients with a pre-operative SARS-CoV-2 diagnosis, mortality was increased in patients having surgery within 0-2 weeks, 3-4 weeks and 5-6 weeks of the diagnosis (odds ratio (95%CI) 4.1 (3.3-4.8), 3.9 (2.6-5.1) and 3.6 (2.0-5.2), respectively). Surgery performed ≥ 7 weeks after SARS-CoV-2 diagnosis was associated with a similar mortality risk to baseline (odds ratio (95%CI) 1.5 (0.9-2.1)). After a ≥ 7 week delay in undertaking surgery following SARS-CoV-2 infection, patients with ongoing symptoms had a higher mortality than patients whose symptoms had resolved or who had been asymptomatic (6.0% (95%CI 3.2-8.7) vs. 2.4% (95%CI 1.4-3.4) vs. 1.3% (95%CI 0.6-2.0), respectively). Where possible, surgery should be delayed for at least 7 weeks following SARS-CoV-2 infection. Patients with ongoing symptoms ≥ 7 weeks from diagnosis may benefit from further delay.

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