KineticDB: a database of protein folding kinetics

We propose here KineticDB, a systematically compiled database of protein folding kinetics, which contains about 90 unique proteins. The main goal of the KineticDB is to provide users with a diverse set of protein folding rates determined experimentally. The search for determinants of protein folding is still in progress, aimed at obtaining a new understanding of the folding process. Comparison with experimental protein folding rates has been the main tool for validation of both theoretical models and empirical relationships during the last 10 years. It is, therefore, necessary to provide a researcher with as much data as possible in a simple and easy-to-use way. At present, the KineticDB contains the results of folding kinetics measurements of single-domain proteins and separate protein domains as well as short peptides without disulfide bonds. It includes data on about 90 unique proteins and many mutants that have been systematically accumulated over the last 10 years and is the largest collection of protein folding kinetic data presented as a database. The KineticDB is available at http://kineticdb.protres.ru/db/index.pl

Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor

The tissue inhibitor of metalloproteinases-3 (TIMP-3) is a major regulator of extracellular matrix turnover and protein shedding by inhibiting different classes of metalloproteinases, including disintegrin metalloproteinases (ADAMs). Tissue bioavailability of TIMP-3 is regulated by the endocytic receptor low-density-lipoprotein receptor-related protein-1 (LRP-1). TIMP-3 plays protective roles in disease. Thus, different approaches have been developed aiming to increase TIMP-3 bioavailability, yet overall effects of increased TIMP-3 in vivo have not been investigated. Herein, by using unbiased mass-spectrometry we demonstrate that TIMP-3-overexpression in HEK293 cells has a dual effect on shedding of transmembrane proteins and turnover of soluble proteins. Several membrane proteins showing reduced shedding are known as ADAM10 substrates, suggesting that exogenous TIMP-3 preferentially inhibits ADAM10 in HEK293 cells. Additionally identified shed membrane proteins may be novel ADAM10 substrate candidates. TIMP-3-overexpression also increased extracellular levels of several soluble proteins, including TIMP-1, MIF and SPARC. Levels of these proteins similarly increased upon LRP-1 inactivation, suggesting that TIMP-3 increases soluble protein levels by competing for their binding to LRP-1 and their subsequent internalization. In conclusion, our study reveals that increased levels of TIMP-3 induce substantial modifications in the cellular secretome and that TIMP-3-based therapies may potentially provoke undesired, dysregulated functions of ADAM10 and LRP-1

The Energy Landscapes of Repeat-Containing Proteins: Topology, Cooperativity, and the Folding Funnels of One-Dimensional Architectures

Repeat-proteins are made up of near repetitions of 20– to 40–amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi–one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete “domain” (the stability and cooperativity of the repeating array) can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (ΔGwater) and the cooperativity of denaturation (m-value), which do not ordinarily apply for globular proteins. We show how the parameters for the “coarse-grained” description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR) repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are “poised” at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions

OneG: A Computational Tool for Predicting Cryptic Intermediates in the Unfolding Kinetics of Proteins under Native Conditions

Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (ΔG) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (ΔGU) and the free energy of exchange (ΔGHX) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ΔGU and ΔGHX of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ΔGU, ΔGU* and residue-specific ΔGHX determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b562 and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.htm

ЕЛЕКТРОТЕПЛОВІ РОЗРАХУНКОВІ МОДЕЛІ ЕЛЕМЕНТІВ КОНСТРУКЦІЇ ТРАНСФОРМАТОРНОГО УСТАТКУВАННЯ

The work provides the review of analytical and empirical calculation methods for calculation of losses and heating of structural elements of oil-cooled power transformer equipment, and also specifies the basic simplifying assumptions. Methodical approaches, developed practical procedures and experience of specified computational investigations with application of finite element system ANSYS are presentedВ работе приведен обзор аналитико-эмпирических методов расчета потерь и нагревов элементов конструкции силового трансформаторного оборудования с масляным охлаждением, указаны основные упрощающие допущения. Представлены методические подходы, разработанные практические процедуры и опыт уточненных численных исследований с применением системы кончено-элементного анализа ANSYSУ роботі приведений огляд аналітико-емпіричних методів розрахунку втрат і нагрівів елементів конструкції силового трансформаторного устаткування з масляним охолодженням, вказані основні спрощуючі допущення. Представлені методичні підходи, розроблені практичні процедури і досвід уточнених чисельних досліджень із застосуванням системи скінчено-елементного аналізу ANSY

ВЕРИФІКАЦІЯ МЕТОДІВ ЕЛЕКТРОТЕПЛОВИХ РОЗРАХУНКІВ ЕЛЕКТРИЧНИХ РЕАКТОРІВ БЕЗ СТАЛІ

Based on the example of the reactor without steel, type ROM-510/26 with electromagnetic shields, verification of analytical and numeral finite-element methods is carried out by the calculation results comparison. For the purpose of corrected analytical calculation, horizontal and vertical shields of the reactor are represented by the system of shortcircuited elements to consider their final dimensions. Calculation is performed as to their inductances, distribution of currents and losses in the shields, magnetic-field and losses in winding, calculation of winding heating by means of the «overheating» empirical method. It is illustrated that analytical calculations correspond to the researches using numeral methods of the electromagnetic and thermal CFD-analysis with sufficient accuracy. For the purpose of practical application in industrial designing of the equipment, the methods with approved and checked measurement results are recommendedНа примере реактора без стали типа РОМ-510/26 с электромагнитными экранами сравнением результатов расчетов проведена верификация аналитических и численных конечно-элементных методов. Для уточненного аналитического расчета горизонтальные и вертикальный экраны реактора для учета их конечных размеров представлены системой короткозамкнутых элементов. Выполняется расчет их индуктивностей, распределения токов и потерь в экранах, магнитного поля и потерь в обмотке, расчет нагрева обмотки эмпирическим методом «перегревов». Показано, что аналитические расчеты с достаточной точностью соответствуют исследованиям численными методами электромагнитного и теплового CFD-анализа. Апробированные и проверенные результатами измерений методы рекомендованы для практического применения при промышленном проектировании оборудованияНа прикладі реактора без сталі типу РОМ-510/26 з електромагнітними екранами порівнянням результатів розрахунків проведена верифікація аналітичних і чисельних скінчено-елементних методів. Для уточненого аналітичного розрахунку горизонтальні і вертикальний екрани реактора для врахування їх кінцевих розмірів представлені системою короткозамкнених елементів. Виконується розрахунок їх індуктивностей, розподілу струмів і втрат в екранах, магнітного поля і втрат в обмотці, розрахунок нагріву обмотки емпіричним методом «перегрівів». Показано, що аналітичні розрахунки з достатньою точністю відповідають дослідженням чисельними методами електромагнітного і теплового CFD-аналізу. Апробовані і перевірені результатами вимірів методи рекомендовані для практичного застосування при промисловому проектуванні устаткуванн