Three-dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (Kd 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed. Published by Wiley-Blackwell. © 2010 The Protein Society

A microscale protein NMR sample screening pipeline

As part of efforts to develop improved methods for NMR protein sample preparation and structure determination, the Northeast Structural Genomics Consortium (NESG) has implemented an NMR screening pipeline for protein target selection, construct optimization, and buffer optimization, incorporating efficient microscale NMR screening of proteins using a micro-cryoprobe. The process is feasible because the newest generation probe requires only small amounts of protein, typically 30–200 μg in 8–35 μl volume. Extensive automation has been made possible by the combination of database tools, mechanization of key process steps, and the use of a micro-cryoprobe that gives excellent data while requiring little optimization and manual setup. In this perspective, we describe the overall process used by the NESG for screening NMR samples as part of a sample optimization process, assessing optimal construct design and solution conditions, as well as for determining protein rotational correlation times in order to assess protein oligomerization states. Database infrastructure has been developed to allow for flexible implementation of new screening protocols and harvesting of the resulting output. The NESG micro NMR screening pipeline has also been used for detergent screening of membrane proteins. Descriptions of the individual steps in the NESG NMR sample design, production, and screening pipeline are presented in the format of a standard operating procedure

Order and disorder in proteins

In contrast to the general view that proteins should have a specific 3D structure in solution for their activity, there are many proteins which do not have a folded “native” structure for a big portion of their sequence. While these intrinsically disordered regions are essential for protein function, they cause problems in efforts for determining the 3D structures for the folded domains. It has been shown that the removal of the disordered domains improved the structure determination success both by X-ray crystallography and by NMR. As part of Northeast Structural Genomics (NESG) effort I worked on identifying the disordered and flexible parts of the protein using Hydrogen/Deuterium Exchange with Mass Spectroscopy (HDX-MS) analysis for construct optimization for high-throughput structure determination. Using this method I also studied human Smad3, which is an important part of the TGF-β-signaling pathway; and provided the first experimental data on structural features of the linker domain. During my training, I also studied human Deleted in Oral Cancer (DOC-1) protein, which was one of the proteins I studied by HDX-MS for construct optimization. We determined the solution structure of the folded region of DOC-1, which was shown to be important in cell-cycle regulation and cancer biology; and I also studied structure-function relations. Additionally, we studied the solution structure of Methionine Sulfoxide Reductase B from Bacillus subtilis, an important protein for reversing oxidative damage in cells, by NMR as a part of methods development studies for NMR for large proteins.Ph. D.Includes bibliographical referencesby Asli Erteki

Spray dried melon seed milk powder: physical, rheological and sensory properties

WOS: 000380102100026PubMed ID: 27407206Melon seed milk (MSM) powder was produced by aiming to get alternative vegetable milk from crushed Kirkagac (Cucumis melo subsp. melo cv. Kirkagac) and Cesme (C. melo subsp. melo cv. Cesme) type melon seeds. MSM was converted to powder form via spray dryer at inlet air temperature of 150 degrees C, air flow rate of 473 l . h(-1), aspiration ratio of 24 m(3) . h(-1) and feed flow rate of 8 ml . min(-1) in order to extend the shelf life and usage area. The moisture content and water activity of samples changed in range of 2.1 to 2.4 % and 0.260 to 0.310, respectively. Bulk densities and the tapped densities of powders were ranged from 340 to 360 kg . m(-3) and 730 and 740 kg . m(-3). MSM powders showed poor flow behavior as determined from Carr Index. The particle densities of powders ranged between and 1069 kg . m(-3). Wettability time of powders was found as 7 s. The Bingham model was the best model fitted to rheological data of MSM beverages. Sensory evaluation test results showed that, the beverage obtained from reconstituted Kirkagac powder achieved the highest score by panelists

Does heparin dosage calculated with ideal body weight reduce blood product use in open heart surgery?

The heparin regimen providing anticoagulation during cardiopulmonary bypass (CPB) is usually adapted to total body weight (TBW). However, this may cause redundant anticoagulation and increase perioperative bleeding, transfusion requirements and reoperation. We compared a heparin regimen based on ideal body weight (IBW) with the traditional regimen. After ethical approval, 100 adults undergoing elective cardiac surgery with CPB were recruited for the prospective, observational study in a tertiary hospital. Prior to CBP an unfractioned heparin dose was adjusted to 300 IU/kg based on TBW (Group TBW, n=50), or IBW (Group IBW, n=50). IBW was calculated using the Lorentz formula. The minimal Activated Coagulation Time (ACT) target value is 400 sec for CPB. Demographic data, cross-clamp and CPB durations, and Hb, Htc, platelet, PT, Aptt and fibrinogen values were similar between the groups (p>0.05). Heparin and total protamine doses were significantly higher in Group IBW (p [Med-Science 2019; 8(1.000): 123-8

Microencapsulation of Extra Virgin Olive Oil by Spray Drying: Effect of Wall Materials Composition, Process Conditions, and Emulsification Method

WOS: 000348130900005The objective of the study was to investigate the microencapsulation of extra virgin olive oil by spray drying to increase its stability and application area. The effects of homogenization rate, pump rate, i.e., flow rate of feeding emulsion and wall materials composition on physical and chemical properties of microencapsulated extra virgin olive oil powder (MEVOP) were evaluated. Maltodextrin (MD) and whey protein isolate (WPI) were used as wall materials and microencapsulation was carried out in a laboratory type spray dryer. The MD:WPI ratio as mixture variable and pump and homogenization rates as process variables were arranged through D-optimal combined design. The optimum wall materials composition and microencapsulation process conditions were determined as follows: 92 % (db) MD, 7 % (db) WPI, and 1 % (db) Tween 20 as wall materials composition and 17,500 rpm and 22 % homogenization and pump rates, respectively. The results showed that the wall materials composition was the most effective independent variables on physical properties in terms of moisture content, water activity, bulk and particle properties of powder as well as microencapsulation efficiency and oxidation stability of MEVOP during converting liquid extra virgin olive oil to powder form. The effects of emulsification methods in terms of rotor-stator and ultrasonic homogenization on physical and chemical properties of MEVOP were also comparatively investigated in this study. The MEVOP produced by ultrasonic homogenization had smaller particle size and lower microencapsulation efficiency than that of rotor-stator homogenization method. But microcapsules obtained by ultrasonic homogenization had better oxidative stability.TUBITAK-TOVAGTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111 O 345]; Ege UniversityEge University; Council of Scientific Research ProjectsEge University [BAP 2010/MUH/011]; EBILTEMEge University [12-BIL-018]The authors acknowledge TUBITAK-TOVAG (Project Number: 111 O 345), Ege University, Council of Scientific Research Projects (Project Number: BAP 2010/MUH/011) and EBILTEM (Project Number: 12-BIL-018) for financial support

Unilateral Antegrade Cerebral Perfusion and Moderate Hypothermia: Assessing Safety With Novel Biomarkers

Background Antegrade cerebral perfusion in aortic surgery is a well-established brain protection method. Open distal anastomosis during aortic surgery has some well-known advantages. Antegrade cerebral perfusion allows repair to some extent of the aortic arch, even in isolated ascending aortic aneurysm. The present study aims to investigate the adequacy of contralateral perfusion with novel oxidative stress parameters during unilateral antegrade cerebral perfusion