Effect of the abolition of intersubunit salt bridges on allosteric protein structural dynamics.

A salt bridge, one of the representative structural factors established by non-covalent interactions, plays a crucial role in stabilizing the structure and regulating the protein function, but its role in dynamic processes has been elusive. Here, to scrutinize the structural and functional roles of the salt bridge in the process of performing the protein function, we investigated the effects of salt bridges on the allosteric structural transition of homodimeric hemoglobin (HbI) by applying time-resolved X-ray solution scattering (TRXSS) to the K30D mutant, in which the interfacial salt bridges of the wild type (WT) are abolished. The TRXSS data of K30D are consistent with the kinetic model that requires one monomer intermediate in addition to three structurally distinct dimer intermediates (I1, I2, and I3) observed in WT and other mutants. The kinetic and structural analyses show that K30D has an accelerated biphasic transition from I2 to I3 by more than nine times compared to WT and lacks significant structural changes in the transition from R-like I2 to T-like I3 observed in WT, unveiling that the loss of the salt bridges interrupts the R-T allosteric transition of HbI. Besides, the correlation between the bimolecular CO recombination rates in K30D, WT, and other mutants reveals that the bimolecular CO recombination is abnormally decelerated in K30D, indicating that the salt bridges also affect the cooperative ligand binding in HbI. These comparisons of the structural dynamics and kinetics of K30D and WT show that the interfacial salt bridges not only assist the physical connection of two subunits but also play a critical role in the global structural signal transduction of one subunit to the other subunit via a series of well-organized structural transitions.

Ultrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering.

Ultrafast motion of molecules, particularly the coherent motion, has been intensively investigated as a key factor guiding the reaction pathways. Recently, X-ray free-electron lasers (XFELs) have been utilized to elucidate the ultrafast motion of molecules. However, the studies on proteins using XFELs have been typically limited to the crystalline phase, and proteins in solution have rarely been investigated. Here we applied femtosecond time-resolved X-ray solution scattering (fs-TRXSS) and a structure refinement method to visualize the ultrafast motion of a protein. We succeeded in revealing detailed ultrafast structural changes of homodimeric hemoglobin involving the coherent motion. In addition to the motion of the protein itself, the time-dependent change of electron density of the hydration shell was tracked. Besides, the analysis on the fs-TRXSS data of myoglobin allows for observing the effect of the oligomeric state on the ultrafast coherent motion.

Performance of Norudia Glycated Albumin Assay on Multiple Analytical Platforms and Comparison to Lucica Assay.

BACKGROUND: The Norudia glycated albumin (GA) assay was evaluated for analytical performance and assay applicability on multiple analytical platforms. METHODS: The evaluation included precision, linearity, reference interval, and comparison with Lucica GA assay. A multicenter study was conducted to compare the results of Norudia GA assay on five kinds of widely used automated clinical chemistry analyzers. RESULTS: Within-laboratory imprecisions for GA% presented 1.3 - 3.3% and 0.8 - 2.6% for low- and high-level control materials, respectively, on different analyzers. GA assay was linear from 20.0 to 680.0 µmol/L of GA. The claimed reference range (12 - 16 GA%) was verified. Norudia GA showed a good GA% correlation with Lucica GA (correlation coefficient 0.999). GA% from each analyzer showed good correlation with the consensus mean of the results of five analyzers (correlation coefficient 0.997 - 0.999). CONCLUSIONS: The Norudia GA assay can successfully be implemented in all the tested platforms, with good GA% correlation.

Mapping the emergence of molecular vibrations mediating bond formation.

Fundamental studies of chemical reactions often consider the molecular dynamics along a reaction coordinate using a calculated or suggested potential energy surface1-5. But fully mapping such dynamics experimentally, by following all nuclear motions in a time-resolved manner-that is, the motions of wavepackets-is challenging and has not yet been realized even for the simple stereotypical bimolecular reaction6-8: A-B + C â†’ A + B-C. Here we track the trajectories of these vibrational wavepackets during photoinduced bond formation of the gold trimer complex [Au(CN)2-]3 in an aqueous monomer solution, using femtosecond X-ray liquidography9-12 with X-ray free-electron lasers13,14. In the complex, which forms when three monomers A, B and C cluster together through non-covalent interactions15,16, the distance between A and B is shorter than that between B and C. Tracking the wavepacket in three-dimensional nuclear coordinates reveals that within the first 60 femtoseconds after photoexcitation, a covalent bond forms between A and B to give A-B + C. The second covalent bond, between B and C, subsequently forms within 360 femtoseconds to give a linear and covalently bonded trimer complex A-B-C. The trimer exhibits harmonic vibrations that we map and unambiguously assign to specific normal modes using only the experimental data. In principle, more intense X-rays could visualize the motion not only of highly scattering atoms such as gold but also of lighter atoms such as carbon and nitrogen, which will open the door to the direct tracking of the atomic motions involved in many chemical reactions.

Effect of Occluded Ligand Migration on the Kinetics and Structural Dynamics of Homodimeric Hemoglobin.

Small molecules such as molecular oxygen, nitric oxide, and carbon monoxide play important roles in life, and many proteins require the transport of small molecules to and from the bulk solvent for their function. Ligand migration within a protein molecule is expected to be closely related to the overall structural changes of the protein, but the detailed and quantitative connection remains elusive. For example, despite numerous studies, how occluded ligand migration affects the kinetics and structural dynamics of the R-T transition remains unclear. To shed light on this issue, we chose homodimeric hemoglobin (HbI) with the I114F mutation (I114F), which is known to interfere with ligand migration between the primary and secondary docking sites, and studied its kinetics and structural dynamics using time-resolved X-ray solution scattering. The kinetic analysis shows that I114F has three structurally distinct intermediates (I1, I2, and I3) as in the wild type (WT), but its geminate CO recombination occurs directly from I1 without the path via I2 observed in WT. Moreover, the structural transitions, which involve ligand migration (the transitions from I1 to I2 and from I3 to the initial state), are decelerated compared to WT. The structural analysis revealed that I114F involves generally smaller structural changes in all three intermediates compared to WT.

Ultrafast structural dynamics of in-cage isomerization of diiodomethane in solution.

Despite extensive studies on the isomer species formed by photodissociation of haloalkanes in solution, the molecular structure of the precursor of the isomer, which is often assumed to be a vibrationally hot isomer formed from the radical pair, and its in-cage isomerization mechanism remain elusive. Here, the structural dynamics of CH2I2 upon 267 nm photoexcitation in methanol were probed with femtosecond X-ray solution scattering at an X-ray free-electron laser. The determined molecular structure of the transiently formed species that converts to the CH2I-I isomer has the I-I distance of 4.17 Å, which is longer than that of the isomer (3.15 Å) by more than 1.0 Å and the mean-squared displacement of 0.45 Å2, which is about 100 times larger than those of typical regular chemical bonds. These unusual structural characteristics are consistent with either a vibrationally hot form of the CH2I-I isomer or the loosely-bound radical pair (CH2I˙⋯I˙).

Diagnostic performance of the Xpert Carba-R assay for active surveillance of rectal carbapenemase-producing organisms in intensive care unit patients.

Background: There are growing concerns regarding the spread of carbapenemase-producing organisms (CPOs) among patients in long-term care facilities (LTCFs) and hospitals in South Korea. We have established a screening protocol for the detection of CPOs in high-risk patients upon admission to intensive care units (ICUs). The diagnostic performance of the Xpert Carba-R assay was compared to that of rectal culture for CPO detection in high-risk patients upon ICU admission. Methods: A total of 408 consecutive rectal swabs were obtained from December 2016 to December 2017. CPO screening was performed using the Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA). When a carbapenemase gene was detected, additional rectal swabs were incubated overnight and inoculated on chromID CARBA medium (bioMérieux, Marcy l'Etoile, France). Bacterial carbapenemase genes, including blaKPC, blaNDM, blaVIM, blaIMP-1, and blaOXA-48, were confirmed by conventional PCR. The diagnostic performance of the Carba-R assay was ascertained based on the culture results. Results: The prevalence of CPO carriage was 7.4% according to the Carba-R assay and 3.7% according to rectal culture. The median Ct values of IMP-1 and KPC were significantly different (35.2 vs. 26.6, P = 0.0143). The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Carba-R assay were 100.0% (95% confidence interval [CI], 78.2-100.0), 96.7% (94.4-98.2), 53.6% (40.4-66.4) and 100.0% (99.0-100.0), respectively. Conclusions: We demonstrated the prevalence of CPO carriage in high-risk patients upon ICU admission and evaluated the diagnostic performance of the Carba-R assay. The combined use of the Xpert Carba-R assay and culture produces rapid and reliable results for the active surveillance of rectal CPO in ICU patients.

Performance evaluation of three rapid antigen tests for the diagnosis of group A Streptococci.

OBJECTIVE: To compare the diagnostic performance of three rapid antigen detection tests (RADTs) for group A Streptococcus (GAS). DESIGN: A hospital-based, cross-sectional, retrospective study. SETTING: A comparative study of rapid diagnostic tests for GAS using clinical specimens in a single institute. PARTICIPANTS: 225 children in the outpatient clinics ofKorea University Guro Hospitalwith suspicious symptoms were subjected to throat swab sampling. A dual-swab applicator was used. Samples were stored at below -70°C in a 10 mL transport tube containing 1 mL liquid Stuart's transport medium. OUTCOME MEASURES: All tests were performed in the laboratory by trained clinical laboratory scientists. Sensitivity, specificity, accuracy and kappa index of three RADTs were compared with the reference PCR test and culture results. RESULTS: Of the 225 patients suspected of having GAS, 67 and 90 were positive for GAS in the culture and PCR tests, respectively. Compared with the reference culture, the sensitivity for GAS was 92.5% (CI 83.4 to 97.5), 71.6% (CI 59.3 to 81.9) and 74.63% (CI 62.5 to 84.4) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively, and the specificity was 97.0% (CI 93.1 to 99.0), 94.6% (CI 90.1 to 97.5) and 92.9% (CI 87.8 to 96.2) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively. Compared with the reference GAS real-time PCR, the sensitivity was 73.3% (CI 62.9 to 82.1), 63.3% (CI 52.5 to 73.2) and 67.8% (CI 57.1 to 77.2) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively, and the specificity was 99.3% (CI 95.9 to 99.9), 100.0% (CI 97.3 to 100.0) and 99.3% (CI 95.9 to 99.9) for careUS Strep A Plus, SD Bioline and BD Veritor, respectively. CONCLUSIONS: The careUS Strep A Plus is a useful test that showed highly comparable results with those of the culture test and superior performances among the three RADTs. The use of RADTs should be encouraged to provide acceptable and fast results using simple equipment.

Protein Structural Dynamics of Wild-Type and Mutant Homodimeric Hemoglobin Studied by Time-Resolved X-Ray Solution Scattering.

The quaternary transition between the relaxed (R) and tense (T) states of heme-binding proteins is a textbook example for the allosteric structural transition. Homodimeric hemoglobin (HbI) from Scapharca inaequivalvis is a useful model system for investigating the allosteric behavior because of the relatively simple quaternary structure. To understand the cooperative transition of HbI, wild-type and mutants of HbI have been studied by using time-resolved X-ray solution scattering (TRXSS), which is sensitive to the conformational changes. Herein, we review the structural dynamics of HbI investigated by TRXSS and compare the results of TRXSS with those of other techniques.

Association between Breastfeeding and Prevalence of Diabetes in Korean Parous Women: The Korea National Health and Nutrition Examination Survey, 2010-2014.

BACKGROUND: It is well known that breastfeeding has a significant impact on the health of mothers and children. With the growing importance of breastfeeding, the present study aimed to investigate the relationship between breastfeeding and the prevalence of diabetes in Korean parous women. METHODS: The data of 5,448 premenopausal parous women aged 20-49 years who agreed to participate in the 5th- 6th Korea National Health and Nutrition Examination Survey were analyzed in this study. Control group included women who had not breastfed. The subjects who had breastfed were classified into three groups based on the duration of breastfeeding: 0-6 months, 6-12 months, and >12 months. The variables included age, body mass index, education level, income, alcohol drinking, smoking, family history of diabetes, use of oral contraceptives, the number of pregnancies, and regular exercise. RESULTS: Among the subjects, the prevalence of diabetes was significantly lower in women who had breastfed compared to those who had not, with an odds ratio of 0.534 (95% confidence interval [CI], 0.289-0.976) in women who breastfed for 0-6 months and 0.575 (95% CI, 0.321-0.990) in women who breastfed for 6-12 months (both P<0.05). CONCLUSION: The present study found a reduced prevalence of diabetes in women who had breastfed compared to those who had not. However, no association between the duration of breastfeeding and the prevalence of diabetes could be found.

The Effects of Weight Control Behaviors among Korean Obese Adults.

BACKGROUND: This study aimed to identify and examine the effects of weight control behaviors correlated with weight loss in obese individuals who attempted to lose weight within the past year. METHODS: In this cross-sectional study, data from 9,461 obese individuals were collected from the fifth and sixth Korean National Health and Nutrition Examination Survey (2010-2014). Three of nine verified methods of weight control suggested in the survey were selected: diet therapy (reduced food intake), exercise therapy (exercise), and medication therapy (prescribed weight loss medications). Participants were divided into one of seven groups (diet therapy alone; exercise therapy alone; drug therapy alone; combined diet and exercise therapy; combined exercise and drug therapy; combined diet and drug therapy; or combined diet, exercise, and drug therapy). Logistic regression analysis was used to determine whether the group that tried to lose weight in the past year had indeed lost weight compared to the group that did not. RESULTS: The odds ratios for weight loss (≥3 but <6 kg vs. ≥6 but <10 kg) for the combined therapies were 2.05 (95% confidence interval, 1.23-3.41) for combined diet and exercise therapy and 5.43 (1.74-16.92) for combined diet, exercise, and drug therapy. CONCLUSION: All levels of weight loss were significantly associated with combined diet and exercise therapy. Weight loss ≥6 kg but <10 kg was significantly associated with combined diet and exercise therapy as well as with combined diet, exercise, and medication therapy among individuals who tried to lose weight in the past year.

Relationship between Marital Status and Metabolic Syndrome in Korean Middle-Aged Women: The Sixth Korea National Health and Nutrition Examination Survey (2013-2014).

BACKGROUND: This study aimed to investigate the relationship between marital status and the incidence of metabolic syndrome in Korean middle-aged women. METHODS: Based on data from the sixth Korea National Health and Nutrition Examination Survey (2013-2014), 3,225 women aged 40-69 years were subjected to the analysis. Marital status was categorized as married, unmarried, separated, widowed, or divorced. The odds ratios (ORs) for metabolic syndrome were calculated based on marital status. After adjustment for age, income level, education level, alcohol intake, smoking status, leisure physical activity, menopause status, daily calories, and fat intake, changes in the OR for metabolic syndrome based on marital status were examined by multivariate logistic regression analysis. RESULTS: The OR for metabolic syndrome in the widowed group to the married group was 4.818 (95% confidence interval [CI], 3.861-6.002; P<0.001) and that after adjustment of age, economic level, education level, alcohol intake, smoking status, physical activity, menopause status, total daily calories, and fat intake was 2.141 (CI, 1.432-3.199; P<0.001), both of which were statistically significant. The OR for metabolic syndrome in the unmarried group to the married group was 0.246 (CI, 0.141-0.431; P<0.001) after adjustment of all components. On the contrary, the ORs of the separated group and the divorced group to the married group were not significant. CONCLUSION: In comparison with the married middle-aged group, the widowed middle-aged group tended to have a higher risk of metabolic syndrome, which is speculated to be related to socioeconomic factors and health behavior.