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

Ambiguities and completeness of SAS data analysis: investigations of apoferritin by SAXS/SANS EID and SEC-SAXS methods

The method of small angle scattering (SAS) is widely used in the field of biophysical research of proteins in aqueous solutions. Obtaining low-resolution structure of proteins is still a highly valuable method despite the advances in high-resolution methods such as X-ray diffraction, cryo-EM etc. SAS offers the unique possibility to obtain structural information under conditions close to those of functional assays, i.e. in solution, without different additives, in the mg/mL concentration range. SAS method has a long history, but there are still many uncertainties related to data treatment. We compared 1D SAS profiles of apoferritin obtained by X-ray diffraction (XRD) and SAS methods. It is shown that SAS curves for X-ray diffraction crystallographic structure of apoferritin differ more significantly than it might be expected due to the resolution of the SAS instrument. Extrapolation to infinite dilution (EID) method does not sufficiently exclude dimerization and oligomerization effects and therefore could not guarantee total absence of dimers account in the final SAS curve. In this study, we show that EID SAXS, EID SANS and SEC-SAXS methods give complementary results and when they are used all together, it allows obtaining the most accurate results and high confidence from SAS data analysis of proteins

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

Folding and form : insights from lattice simulations

Monte Carlo simulations of a Miyazawa-Jernigan lattice-polymer model indicate that, depending on the native structure's geometry, the model exhibits two broad classes of folding mechanisms for two-state folders. Folding to native structures of low contact order is driven by backbone distance and is characterized by a progressive accumulation of structure towards the native fold. By contrast, folding to high contact order targets is dominated by intermediate stage contacts not present in the native fold, yielding a more cooperative folding process