From Microstates to Macrostates in the Conformational Dynamics of GroEL: A Single-Molecule Förster Resonance Energy Transfer Study.

The chaperonin GroEL is a multisubunit molecular machine that assists in protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. Here, we report single-molecule Förster resonance energy transfer measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the cochaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-ligated conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis. These coinciding results from bulk measurements for an entire ring and single-molecule measurements for a single subunit provide new evidence for the concerted allosteric transition of all seven subunits.

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase.

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Substrates Modulate Charge-Reorganization Allosteric Effects in Protein-Protein Association.

Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.

Maternal history of recurrent pregnancy loss and long-term risk of thromboembolic events.

BACKGROUND: Recurrent pregnancy loss is a multifactorial disorder with potential underlying maternal hypercoagulability. OBJECTIVE: We aimed to investigate whether a history of recurrent pregnancy loss poses an independent risk for future maternal long-term thromboembolic morbidity. STUDY DESIGN: A population-based study compared the incidence of long-term thromboembolic morbidity in a cohort of women with and without a history of recurrent pregnancy loss. Data were collected from two databases that were cross-linked and merged: a computerized hospitalization database and a computerized obstetrics and gynecology perinatal database. Deliveries occurred between the years 1991-2017. The risk for long-term thromboembolic morbidity was based on the hospital's database and a pre-defined set of ICD codes of thromboembolic related hospitalizations. A Kaplan-Meier survival curve was constructed to compare the cumulative incidence of significant thromboembolic morbidity in the exposed and unexposed groups, and a Cox proportional hazards model, to control for confounders. RESULTS: During the study period, 123,791 parturients met the inclusion criteria; 6.7 % n = 8247 of which experienced previous recurrent pregnancy loss-the exposed group. The rate of thromboembolic related hospitalizations was almost double in exposed women as compared to non-exposed 1.1 % vs. 0.6 %, OR = 1.8; 95 % 1.4-2.2; p < 0.001). Cumulative thromboembolic event incidence, as depicted in the survival curves, was also significantly higher among patients with recurrent pregnancy loss history (Log rank p < 0.001). The Cox model confirmed recurrent pregnancy loss history to be an independent risk factor for later maternal thromboembolic morbidity, while controlling for maternal age, known thrombophilia, ethnicity, smoking, hypertension, diabetes, and fertility treatment (aHR 1.27, 95 %CI 1.02-1.59, p=0.034). CONCLUSION: A history of recurrent pregnancy loss is an independent risk factor for long-term thromboembolic morbidity, even in the absence of known maternal thrombophilia.

Prevalence and risk factors for urinary tract infection following stress urinary incontinence surgery with two midurethral sling procedures.

OBJECTIVE: To compare prevalence and risk factors for urinary tract infection (UTI) following midurethral sling surgery with either the GYNECARE ABBREVO Continence System (Ethicon, Somerville, NJ, USA) or a standard transobturator. METHODS: A retrospective cohort study was conducted among women who underwent midurethral sling surgery to treat stress urinary incontinence at a single tertiary hospital in Israel between January 1, 2014, and August 11, 2015. Data were retrieved from medical records. The diagnosis of UTI was based on a positive urine culture result. RESULTS: Of 178 patients included, 30 (16.9%) underwent the ABBREVO procedure and 148 (83.1%) underwent the standard transobturator procedure. The mean ± SD BMI at the time of surgery was 28.1 ± 4.5 for the standard transobturator procedures and 30.7 ± 15.2 for the ABBREVO procedures. Women who underwent the ABBREVO procedure had an increased duration of hospitalization (P=0.004), and higher rates of concomitant anterior colporrhaphy procedures (P=0.009) and concomitant hysterectomy (P=0.009). Only 38 (21.3%) women developed UTIs within 12 months of surgery (seven in the ABBREVO procedure group and 31 in the standard transobturator procedure group). No statistically significant between-group differences were found for the risk of UTI or for urinary tract adverse events, such as recurrent UTI and the need for rehospitalization. CONCLUSIONS: The two procedures were comparable in terms of prevalence and risk factors for UTI during the postoperative period.

Prevalence and risk factors for urinary tract infection up to one year following midurethral sling incontinence surgery.

OBJECTIVE: To investigate the prevalence and risk factors of urinary tract infection (UTI) one year following sub-midurethral sling (SMUS) incontinence surgery in a university affiliated medical center in southern Israel. METHODS: A retrospective cohort study was conducted to identify and characterize patients who suffered UTI within a year following SMUS surgery. The study population comprised of all patients who underwent a SMUS surgery between the years 2014 and 2015. Demographic and clinical data were retrieved from the patients' medical records, and a comparison between patients with and without a positive urine culture was performed. RESULTS: During the study period, there were 178 SMUS surgeries. Urine culture positive UTI was noted in 21% (38 patients) within the first year following surgery. The mean age and BMI of patients complicated with UTI was 64.8 and 29.1, respectively. The most common pathogen found in urine culture was E. coli that accounted for 55% of all UTIs. When comparing patients with and without UTI, no significant difference was noted in the pre- and intra-operative characteristics. However, duration of hospitalization and readmissions in the first year following surgery, were significantly associated with the risk of UTI (p < 0.026 and p < 0.003, respectively). CONCLUSIONS: Approximately one in every five women undergoing a SMUS operation in our population will suffer from UTI within a year from surgery. A significant association was found between the duration of hospitalization and readmissions in the first postoperative year and suffering from UTI.

Native tissue repair for central compartment prolapse: a narrative review.

Central descent due to a level 1 defect is a main component in pelvic organ prolapse (POP) reconstructive surgery, whether for symptomatic apical prolapse or for the prolapse repair of other compartments. A recent growth in the rate of native tissue repair procedures for POP, following the US Food and Drug Administration (FDA) warnings regarding the safety and efficacy of synthetic meshes, requires a re-evaluation of these procedures. The safety, efficacy, and determination of the optimal surgical approach should be the center of attention. Functional outcome measures and patient-centered results have lately gained importance and received focus. A comprehensive literature review was performed to evaluate objective and subjective outcomes of apical prolapse native tissue repair, with a special focus on studies reporting impact on patients' functional outcomes, quality of life, and satisfaction. We performed a MEDLINE search for articles in the English language by using the following key words: apical prolapse, sacrospinous ligament fixation, uterosacral ligament suspension, sacral colpopexy, McCall culdoplasty, iliococcygeus vaginal fixation, and functional outcomes. We reviewed references as well. Despite a prominent shortage of studies reporting standardized prospective outcomes for native tissue repair interventions, we noted a high rate of safety and efficacy, with a low complication rate for most procedures and low recurrence or re-treatment rates. The objective and subjective results of different procedures are reviewed. Functional outcomes of native tissue repair procedures have not been studied sufficiently, though existing data present those procedures as favorable and not categorically inferior to sacrocolpopexy. Apical compartment prolapse repair using native tissue is not a compromise. Functional outcomes of native tissue repair procedures are favorable, have a high rate of success, improve women's quality of life (QoL), and result in high rates of patient satisfaction. This subject requires further long-term, standardized prospective studies following the International Continence Society/International Urogynecologists Association guidelines for surgical outcomes report, with the focus on patient-centered functional outcomes.