Chaperonin GroEL hydrolyses ortho-nitrophenyl ß-galactoside.

We serendipitously found that chaperonin GroEL can hydrolyze ortho-nitrophenyl ß-galactoside (ONPG), a well-known substrate of the enzyme ß-galactosidase. The ONPG hydrolysis by GroEL follows typical enzyme kinetics. Our experiments and molecular docking studies suggest ONPG binding at the ATP binding site of GroEL.

Modified Protein-Water Interactions in CHARMM36m for Thermodynamics and Kinetics of Proteins in Dilute and Crowded Solutions.

Proper balance between protein-protein and protein-water interactions is vital for atomistic molecular dynamics (MD) simulations of globular proteins as well as intrinsically disordered proteins (IDPs). The overestimation of protein-protein interactions tends to make IDPs more compact than those in experiments. Likewise, multiple proteins in crowded solutions are aggregated with each other too strongly. To optimize the balance, Lennard-Jones (LJ) interactions between protein and water are often increased about 10% (with a scaling parameter, λ = 1.1) from the existing force fields. Here, we explore the optimal scaling parameter of protein-water LJ interactions for CHARMM36m in conjunction with the modified TIP3P water model, by performing enhanced sampling MD simulations of several peptides in dilute solutions and conventional MD simulations of globular proteins in dilute and crowded solutions. In our simulations, 10% increase of protein-water LJ interaction for the CHARMM36m cannot maintain stability of a small helical peptide, (AAQAA)3 in a dilute solution and only a small modification of protein-water LJ interaction up to the 3% increase (λ = 1.03) is allowed. The modified protein-water interactions are applicable to other peptides and globular proteins in dilute solutions without changing thermodynamic properties from the original CHARMM36m. However, it has a great impact on the diffusive properties of proteins in crowded solutions, avoiding the formation of too sticky protein-protein interactions.

Elucidation of interactions regulating conformational stability and dynamics of SARS-CoV-2 S-protein.

The ongoing COVID-19 pandemic caused by the new coronavirus, SARS-CoV-2, calls for urgent developments of vaccines and antiviral drugs. The spike protein of SARS-CoV-2 (S-protein), which consists of trimeric polypeptide chains with glycosylated residues on the surface, triggers the virus entry into a host cell. Extensive structural and functional studies on this protein have rapidly advanced our understanding of the S-protein structure at atomic resolutions, although most of these structural studies overlook the effect of glycans attached to the S-protein on the conformational stability and functional motions between the inactive down and active up forms. Here, we performed all-atom molecular dynamics simulations of both down and up forms of a fully glycosylated S-protein in solution as well as targeted molecular dynamics simulations between them to elucidate key interdomain interactions for stabilizing each form and inducing the large-scale conformational transitions. The residue-level interaction analysis of the simulation trajectories detects distinct amino acid residues and N-glycans as determinants on conformational stability of each form. During the conformational transitions between them, interdomain interactions mediated by glycosylated residues are switched to play key roles on the stabilization of another form. Electrostatic interactions, as well as hydrogen bonds between the three receptor binding domains, work as driving forces to initiate the conformational transitions toward the active form. This study sheds light on the mechanisms underlying conformational stability and functional motions of the S-protein, which are relevant for vaccine and antiviral drug developments.

Vibrational Energy Transfer from Heme through Atomic Contacts in Proteins.

A pathway of vibrational energy flow in myoglobin was studied by time-resolved anti-Stokes ultraviolet resonance Raman spectroscopy combined with site-directed mutagenesis. Our previous study suggested that atomic contacts in proteins provide the dominant pathway for energy transfer while covalent bonds do not. In the present study, we directly examined the contributions of covalent bonds and atomic contacts to the pathway of vibrational energy flow by comparing the anti-Stokes resonance Raman spectra of two myoglobin mutants: one lacked a covalent bond between heme and the polypeptide chain, and the other retained the intact bond. The two mutants showed no significant difference in temporal changes in the anti-Stokes Raman intensities of the tryptophan bands, implying that the dominant channel of vibrational energy transfer is not through the covalent bond but rather through van der Waals atomic contacts between heme and the protein moiety. The obtained insights contribute to our general understanding of energy transfer in the condensed phase.

A Pseudohypervalent Sulfur Intermediate as an Oxidative Protective Mechanism in the Archaea Peroxiredoxin Enzyme ApTPx.

Peroxiredoxins (Prxs) are a ubiquitous class of enzymes that have central roles in a number of important physiological processes. Using a multiscale computational approach, we have investigated the mechanism by which the active-site cysteine (Cys50) in the typical 2-Cys Prx from Archaea (ApTPx) is oxidized by H2O2 to sulfenic acid. In addition, its further oxidation to give a sulfinic acid and its possible alternate intramolecular reaction to form an experimentally proposed hypervalent sulfurane were examined. Oxidation of Cys50 by H2O2 to give the sulfenic acid intermediate occurs in one step with a barrier of 82.1 kJ mol-1. A two-step pathway is proposed with a very low barrier of 16.5 kJ mol-1 by which it can subsequently react with an adjacent histidyl (His42) to form the pseudohypervalent sulfurane. This pathway also involves an adjacent aspartyl (Asp45), which helps alternate the protonation state of His42. The sulfurane's Cys50S···NδHis42 interaction was characterized using QTAIM, NCI, and NBO analyses and found to be a noncovalent interaction. Notably, this bond helps orient the Cys50SOH moiety such that it is less susceptible to oxidation by H2O2 to sulfinic acid. Significantly, sulfurane formation is energetically favored to further H2O2 oxidation of Cys50SOH to a sulfinic acid, providing a mechanism by which the active-site Cys50 is protected against overoxidation.

Factors Associated with Low Flow in Aortic Valve Stenosis.

BACKGROUND: Low-flow low-gradient aortic stenosis (AS) is a predictor of worse outcome compared with normal-flow AS. Although depressed left ventricular ejection fraction (LVEF) is associated with low flow, there is less evidence to support the role of other indices of cardiac structure and function. METHODS: Clinical and echocardiographic data from patients with native AS and valve areas ≤ 1.0 cm(2) were retrospectively analyzed to identify characteristics that are associated with low-flow low-gradient AS. RESULTS: In total, 941 patients were included. On multivariate analysis, factors independently associated with low flow (stroke volume index < 35 mL/m(2)) included worse right ventricular systolic function, atrial fibrillation, lower LVEF, and higher left ventricular mass, with moderate or severe mitral regurgitation independently associated with low flow in the 694 patients (74%) with preserved LVEFs. CONCLUSIONS: Right ventricular dysfunction and atrial fibrillation are independently associated with low-flow low-gradient AS, while moderate or severe MR is independently associated with low flow in patients with preserved LVEF. These associations with low flow in AS are clinically important to recognize, to avoid underestimation of AS severity.

Computational investigations on the catalytic mechanism of maleate isomerase: the role of the active site cysteine residues.

The maleate isomerase (MI) catalysed isomerization of maleate to fumarate has been investigated using a wide range of computational modelling techniques, including small model DFT calculations, QM-cluster approach, quantum mechanical/molecular mechanical approach (QM/MM in the ONIOM formalism) and molecular dynamics simulations. Several fundamental questions regarding the mechanism were answered in detail, such as the activation and stabilization of the catalytic Cys in a rather hydrophobic active site. The two previously proposed mechanisms were considered, where either enediolate or succinyl-Cys intermediate forms. Small model calculations as well as an ONIOM-based approach suggest that an enediolate intermediate is too unstable. Furthermore, the formation of succinyl-Cys intermediate via the nucleophilic attack of Cys76(-) on the substrate C2 (as proposed experimentally) was found to be energetically unfeasible in both QM-cluster and ONIOM approaches. Instead, our results show that Cys194, upon activation via the substrate, acts as a nucleophile and Cys76 acts as an acid/base catalyst, forming a succinyl-Cys intermediate in a concerted fashion. Indeed, the calculated PA of Cys76 is always higher than that of Cys194 before or upon substrate binding in the active site. Furthermore, the mechanism proceeds via multiple steps by substrate rotation around C2-C3 with the assistance of the now negatively charged Cys76, leading to the formation of fumarate. Finally, our calculated barrier is in good agreement with experiment. These findings represent a novel mechanism in the racemase superfamily.

A molecular dynamics and quantum mechanics/molecular mechanics study of the catalytic reductase mechanism of methionine sulfoxide reductase A: formation and reduction of a sulfenic acid.

The catalytic mechanism of MsrA in Mycobacterium tuberculosis, in which S-methionine sulfoxide (Met-O) is reduced to methionine (Met), has been investigated using docking, molecular dynamics (MD) simulations, and ONIOM (quantum mechanics/molecular mechanics) methods. In addition, the roles of specific active site residues, including an aspartyl (Asp87) near the recycling cysteine, tyrosyls (Tyr44 and Tyr92), and glutamyl (Glu52), have been examined, as well as the general effects of the protein and active site on the nature and properties of mechanistic intermediates. The mechanism is initiated by the transfer of a proton from the catalytic cysteine's thiol (Cys13SH) via a bridging water to the R group carboxylate of Glu52. The now anionic sulfur of Cys13 nucleophilically attacks the substrate's sulfur with concomitant transfer of a proton from Glu52 to the sulfoxide oxygen, generating a sulfurane. The active site enhances the proton affinity of the sulfurane oxygen, which can readily accept a proton from the phenolic hydroxyls of Tyr44 or Tyr92 to give a sulfonium cation. Subsequently, Asp87 and the recycling cysteine (Cys154) can facilitate nucleophilic attack of a solvent water at the Cys13S center of the sulfonium to give a sulfenic acid (Cys13SOH) and Met. For the subsequent reduction of Cys13SOH with intramolecular disulfide bond formation, Asp87 can help facilitate nucleophilic attack of Cys154S at the sulfur of Cys13SOH by deprotonating its thiol. This reduction is found likely to occur readily upon suitable positioning of the active site hydrogen bond network and the sulfur centers of both Cys13 and Cys154. The calculated rate-limiting barrier is in good agreement with experiment.

Assessment of the American Society of Echocardiography-European Association of Echocardiography guidelines for diastolic function in patients with depressed ejection fraction: an echocardiographic and invasive haemodynamic study.

AIMS: There is controversy surrounding the accuracy of echo-Doppler variables, including early mitral inflow/mitral annular velocity (E/e'), for estimating left ventricular filling pressure (LVFP) in patients with depressed ejection fraction (EF < 50%). METHODS AND RESULTS: The American Society of Echocardiography-European Association of Echocardiography (ASE-EAE) algorithm for diastolic function in depressed LVEF was retrospectively applied to a database of patients who underwent echocardiography ≤20 min of cardiac catheterization. LV pre-atrial contraction pressure (pre-A) ≥15 mmHg was elevated. Of 62 patients studied, the mean age was 53.6 ± 10.6 years and the mean LVEF was 27.2 ± 11.8%. The correlations of E/e' (R = 0.43, P = 0.0005) and E (R = 0.39, P = 0.002) with LV pre-A were modest, compared with pulmonary artery pressure (PAP, R = 0.69, P = 0.0006), E/late mitral (A) velocity (R = 0.52, P < 0.0001), and mitral deceleration time (DT, R = -0.51, P < 0.0001). Using the ASE-ESE algorithm starting with E/A, E, and DT, 54 of 62 patients were accurately classified to predict LV pre-A >15 or <15 mmHg (sensitivity = 84%, specificity = 80%, area under the curve = 0.86, P < 0.001). The 6 of 6 patients with E/A < 1 and E < 50 and the 14 of 15 (93%) patients with E/A> 2 and DT < 150 were correctly classified as having normal and elevated LVFP, respectively, while 34 of 41 (83%) patients with E/A = 1-2 or E/A<1 and E>50 cm/s were correctly classified using the addition of E/e' and PAP. CONCLUSION: This retrospective study shows that in this population with depressed LVEF, no single echo-Doppler variable had high accuracy for predicting LV pre-A ≥15 mmHg. However, the ASE-EAE algorithm using multiple variables predicted LVFP with good accuracy, superior to any single echo-Doppler variable alone.

New, simple echocardiographic indexes for the estimation of filling pressure in patients with cardiac disease and preserved left ventricular ejection fraction.

BACKGROUND: There are few data on echocardiographic indexes incorporating peak mitral inflow velocity (E), left atrial volume index (LAVi), and pulmonary artery pressure (PAP) for estimation of left ventricular (LV) filling pressure in patients with preserved LV ejection fraction (EF ≥ 50%). METHODS: Patients underwent echocardiography ≤20 minutes of cardiac catheterization. Echocardiographic variables were compared to invasively measured LV end-diastolic pressure (LVEDP). RESULTS: Of the 122 patients, 67 (55%) were women, the mean age was 55 ± 9 years, the mean left ventricular ejection fraction (LVEF) was 61 ± 6%, 107 (88%) were hypertensive, and 79 (65%) had significant coronary artery disease at catheterization. E/Ea correlated with LVEDP (R = 0.68, P < 0.0001), compared to PAP (R = 0.53, P < 0.001), peak E velocity (R = 0.48, P < 0.001), and LAVi (R = 0.48, P < 0.001). E/Ea > 12 had 75% sensitivity and 78% specificity for LVEDP ≥ 20 mmHg (area under curve (AUC) = 0.79, P < 0.0001), compared with (PAP + LAVi)/2 > 30 (sensitivity = 72%, specificity = 80%, AUC = 0.84, P < 0.001) and (E + LAVi)/2 > 57 (sensitivity = 73% and specificity = 81%, AUC = 0.82, P < 0.001) (P = NS). E <60 cm/sec had 94% negative, and E>90 cm/sec had 96% positive, predictive value for LVEDP ≥ 20 mmHg. (E + LAVi)/2 added incrementally to E/Ea when E/Ea was in the gray zone. CONCLUSIONS: New, simple echocardiographic equations, (E + LAVi)/2 and (PAP + LAVi)/2, have comparable accuracy to E/Ea for LVEDP estimation in patients with cardiac disease and preserved LVEF, and (E + LAVi)/2 added incrementally to E/Ea alone when E/Ea was in the gray zone. Peak E velocity alone had high negative and positive predictive value for elevated LVEDP in this population. These simple echocardiographic variables could be used-in isolation or with E/Ea-in patients with cardiac disease and preserved LVEF for the diagnosis of diastolic heart failure.

Do additional echocardiographic variables increase the accuracy of E/e' for predicting left ventricular filling pressure in normal ejection fraction? An echocardiographic and invasive hemodynamic study.

BACKGROUND: There are few data on adding left atrial volume index (LAVi) or pulmonary artery systolic pressure (PAP) to the ratio of early mitral inflow to mitral annular velocity (E/e') for the estimation of left ventricular (LV) filling pressure in patients with preserved LV ejection fractions (LVEFs) (>50%). METHODS: Patients underwent echocardiography within 20 minutes of cardiac catheterization. Echocardiographic variables were compared with invasively measured LV preatrial contraction pressure (pre-A). RESULTS: Of the 122 patients studied (mean age, 55 +/- 9 years; mean LVEF, 61 +/- 6%), 67 (55%) were women, 108 (88%) had hypertension, and 79 (65%) had significant coronary artery disease at catheterization. E/e' was significantly correlated with pre-A (R = 0.63, P < .0001) compared with LAVi (R = 0.49, P < .001) and PAP (R = 0.48, P < .001). E/e' > 13 had sensitivity of 70% and specificity of 93% (area under the curve [AUC], 0.82; P < .0001), LAVi > 31 mL/m2 had sensitivity of 78% and specificity of 76% (AUC, 0.80, P < .001), and PAP > 28 mm Hg had sensitivity of 80% and specificity of 64% for pre-A > 15 mm Hg (AUC, 0.77, P < .001). Adding LAVi >31 mL/m2 for E/e' = 8 to 13 significantly increased the accuracy of E/e' > 13 alone (sensitivity, 87%; specificity, 88%; AUC, 0.89; P = .01 for comparison). However, adding PAP > 28 mm Hg for E/e' = 8 to 13 did not significantly increase the accuracy of E/e' > 13 alone (AUC, 0.82; sensitivity, 82%; specificity, 72%; P = NS for comparison). CONCLUSIONS: In patients with preserved LVEFs, adding LAVi > 31 mL/m2 to E/e' (when E/e' was in the gray zone, but not when E/e' was >13) significantly increased the accuracy of E/e' alone for the estimation of LV filling pressure. These data support the notion of using several, rather than any single, Doppler echocardiographic parameter for the accurate assessment of LV diastolic function.

In vitro inhibition of platelet aggregation in response to increasing concentrations of tirofiban in patients with significant renal insufficiency.

BACKGROUND: Patients with impaired renal function are a growing subset at higher risk for cardiovascular complications due to vasculopathic state, inducing accelerated atherosclerosis and arteriosclerosis. These patients are at increased risk for complications after coronary interventions, especially major bleeding events. As a result, this at-risk population of patients has not been well studied in most of the major clinical trials evaluating coronary interventions. Of particular interest is the optimal dosing of glycoprotein IIb-IIIa inhibitors in the setting of acute myocardial infarction. In this study, we attempted to find the in vitro concentration of tirofiban required to inhibit platelet aggregation to <10% in patients with moderate to severe renal insufficiency. METHODS: A total of 21 patients were divided into two groups based on estimated creatinine clearance (group 1 <46ml/min; group 2 >46ml/min). Platelet-rich plasma from each subject was then incubated in vitro with increasing concentrations of tirofiban (25, 37.5, and 50ng/ml), and light transmission aggregometry assay was used to assess the degree of platelet aggregation in response to adenosine diphosphate (ADP). RESULTS: Patients in group 1 had a baseline platelet aggregation of 45%, which decreased to 10% at a 25.0ng/ml concentration of tirofiban; the effect was enhanced to a platelet aggregation of <5% at higher doses. In contrast, subjects in group 2 with creatinine clearance >or=46ml/min had an average platelet aggregation inhibition of 12% with 50ng/ml of tirofiban.We found a significant decrease in platelet aggregation in group 2 at 25, 37.5, and 50ng/ml of tirofiban (p<0.05) in comparison with group 1. CONCLUSIONS: Our data indicate that patients with moderate to severe renal dysfunction suppress their platelet aggregation to <10% with 25ng/ml of tirofiban, one-third of the standard effective dose for patients with normal renal function.We suggest further clinical trials to define an objective means to calculate proper renal dosing of glycoprotein IIb-IIIa inhibitors in these patients to prevent potentially fatal complication of major hemorrhagic events.

Hypertrophic obstructive cardiomyopathy-alcohol septal ablation vs. myectomy: a meta-analysis.

AIMS: Our purpose is to conduct a meta-analysis of all published studies comparing alcohol septal ablation (ASA) and myectomy (MM) for drug refractory hypertrophic obstructive cardiomyopathy (HOCM). Alcohol septal ablation is a less invasive alternative to MM for relief of symptoms in patients with drug refractory HOCM. METHODS AND RESULTS: An extensive search of PubMed identified five non-randomized studies comparing ASA and MM. Of 351 patients studied, 183 underwent ASA and 168 underwent MM. Patients undergoing ASA were older (mean age 54.4 +/- 6.3 vs. 45.0 +/- 4.4 years, P = 0.02). All patients were in New York Heart Association (NYHA) class II-IV. Baseline left ventricular outflow tract (LVOT) gradient was comparable (81.4 +/- 14.3 mmHg in ASA vs. 77.4 +/- 15.5 mmHg in MM, P = 0.2). Although resting LVOT gradient after septal reduction therapy in both groups was <20 mmHg at follow-up, patients undergoing MM had lower LVOT gradient (18.2 +/- 6.7 vs. 10.8 +/- 6.3 mmHg, P < 0.001). Patients in both groups had comparable improvement in NYHA class (1.5 +/- 0.3 in ASA vs. 1.3 +/- 0.2, P = 0.2) at follow-up. A higher percentage of patients undergoing ASA required permanent pacemaker (PPM) implantation for complete heart block (18.4 +/- 7.9 vs. 3.3 +/- 3.9%, P = 0.04). There was no significant in-hospital mortality difference between the two groups. CONCLUSION: Alcohol septal ablation and MM provide significant reduction in LVOT gradient and NYHA functional class on mid-term follow-up. A higher percentage of patients required PPM after ASA. Randomized trials are needed to confirm current findings.

Role of inflammation in initiation and perpetuation of atrial fibrillation: a systematic review of the published data.

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. Recent studies have indicated that inflammation might play a significant role in the initiation, maintenance, and perpetuation of AF. Inflammatory markers such as interleukin-6 and C-reactive protein are elevated in AF and correlate to longer duration of AF, success of cardioversion, and thrombogenesis. Furthermore, the inflammatory process might be modulated by the use of statins, angiotensin-converting enzyme inhibitors, or glucocorticoids. The purpose of this study is to analyze the current published reports on the relationship between inflammation and AF and the potential therapeutic options available to modulate the inflammatory milieu in AF.

Comprehensive imaging including three-dimensional echocardiography of an infected, ruptured sinus of valsalva aneurysm.

A 30-year-old man presented with fevers and fatigue. Blood cultures grew Streptococcus mitis in 4/4 bottles. Transthoracic three-dimensional echocardiography revealed an anterior coronary sinus of Valsalva (SOV) aneurysm with fistula formation into the right ventricle with vegetation on the fistulous tract, and a bicuspid aortic valve without vegetation. Transesophageal echocardiography confirmed these findings. After parenteral antibiotic treatment, the patient went for successful repair of the fistula, with the imaging findings confirmed at surgery. This case represents a rare complication of a ruptured SOV aneurysm, with excellent delineation of cardiac anatomy using transthoracic three-dimensional echocardiography.

The obesity paradox: fact or fiction?

Although the adverse health consequences of obesity in the general population have been well documented, recent evidence suggests that obesity is associated with better outcomes in patients with heart failure (HF). Studies of patients with HF that specifically examined the impact of body mass index (BMI) on outcomes have suggested the existence of an "obesity paradox." However, closer examination of these studies raises important questions on the validity of the paradox. First, the diagnosis of HF in obese patients, particularly when made using clinical variables, may not be accurate; the obese patients in these studies may actually be "healthier" than their nonobese comparators. Second, the deleterious effects of cachexia, rather than the salutary ones of obesity, are likely the main reason for the inverse correlation between BMI and HF outcome, especially once the underlying biologic mechanisms behind cachexia and obesity in patients with HF are considered. Furthermore, few studies have specifically examined the more severely obese population (BMI >35 kg/m(2)) when assessing outcomes, and those that have suggest that severely obese patients may have worse outcomes than patients with normal weights or those who are mildly obese. Therefore, a "U-shaped" outcome curve according to BMI for patients with HF may actually exist, in which mortality is greatest in cachectic patients; lower in normal, overweight, and mildly obese patients; but higher again in more severely obese patients. Further prospective studies assessing the impact of more marked degrees of obesity on outcomes in patients with HF are needed to more conclusively determine whether the obesity paradox truly exists.

Visceral pericardial hemangioma: unusual location for a rare cardiac tumor.

A 41-year-old man presented with chest pain and a positive electrocardiographic stress treadmill test. Coronary angiography revealed no significant coronary disease, but suggested a mass posterior to the heart. Transthoracic and transesophageal echocardiography revealed a smooth-surfaced mass in the inferoposterior atrioventricular groove, with color Doppler evidence of vascular formation within the mass. Magnetic resonance imaging confirmed the presence of the tumor, without extension into adjacent cardiac chambers or pericardial effusion. At operation, a 4.6- x 3.0-cm tumor attached to the visceral pericardium was excised. Pathologic section resulted in a diagnosis of hemangioma. Hemangiomas account for 2% to 5% of benign cardiac tumors, arising from the cardiac ventricles, atria, valves, and, rarely, the epicardium/pericardium. This case illustrates a very rare location for an unusual benign cardiac tumor. The patient recovered after operation without complication.