Screening for Novel Calcium-Binding Proteins that Regulate Cardiac Hypertrophy: CIB1 as an Example
Calcium-binding proteins have a crucial function in the regulation of cardiac contractility as well as in the regulation of cardiac signal-transduction. Because they sense calcium concentrations and at the same time bind specific signaling molecules, some of these proteins are critically involved in the establishment of signaling microdomains, which are insulated from the large cytosolic calcium fluctuations involved in cardiac excitation–contraction coupling. In this regard, we have recently identified the calcium-binding protein CIB1 as an important regulator of pathological cardiac hypertrophy and transition to he...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

RAGE Splicing Variants in Mammals
The receptor for advanced glycation end products (RAGE) is a multiligand receptor of environmental stressors which plays key roles in pathophysiological processes, including immune/inflammatory disorders, Alzheimer’s disease, diabetic arteriosclerosis, tumorigenesis, and metastasis. Besides the full-length RAGE protein in humans nearly 20 natural occurring RAGE splicing variants were described on mRNA and protein level. These naturally occurring isoforms are characterized by either N-terminally or C-terminally truncations and are discussed as possible regulators of the full-length RAGE receptor either by competitive ...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

RAGE-Mediated Cell Signaling
RAGE (receptor for advanced glycation end products) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of transmembrane proteins. RAGE binds AGEs (advanced glycation end products), HMGB1 (high-mobility group box-1; also designated as amphoterin), members of the S100 protein family, glycosaminoglycans and amyloid β peptides. Recent studies using tools of structural biology have started to unravel common molecular patterns in the diverse set of ligands recognized by RAGE. The distal Ig domain (V1 domain) of RAGE has a positively charged patch, the geometry of which fits to anionic surfaces display...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Phage Display Selection of Peptides that Target Calcium-Binding Proteins
Phage display allows to rapidly identify peptide sequences with binding affinity towards target proteins, for example, calcium-binding proteins (CBPs). Phage technology allows screening of 109 or more independent peptide sequences and can identify CBP binding peptides within 2 weeks. Adjusting of screening conditions allows selecting CBPs binding peptides that are either calcium-dependent or independent. Obtained peptide sequences can be used to identify CBP target proteins based on sequence homology or to quickly obtain peptide-based CBP inhibitors to modulate CBP-target interactions. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Measuring Binding of S1 Proteins to RAGE by Surface Plasmon Resonance
Surface plasmon resonance (SPR) is a label-free biophysical method that allows to measure the binding parameters (ka, kd, KD) of the interaction between two molecules. In this method, one protein/molecule (ligand) is immobilized on the surface of a sensor chip, while the other molecule (analyte) is in solution. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Biochemical and Immunological Detection of Physical Interactions Between Penta-EF-Hand Protein ALG-2 and Its Binding Partners
Many nonenzymatic cellular proteins exert their functions by interacting with other proteins or ­macromolecules. Analysis of the physical interactions of proteins is an important step to understand their functions, and the information obtained is helpful for predicting the roles of the proteins in cells. Here we describe three biochemical and immunological methods for the detection of interactions between ALG-2 (a penta-EF-hand Ca2+-binding protein, also known as PDCD6) and its target proteins: (1) glutathione-S-transferase (GST) pulldown assay, (2) co-immunoprecipitation assay, and (3) Far Western blot analysis using ...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

NMR Studies of the Interaction of Calmodulin with IQ Motif Peptides
We describe here methods for the robust expression and purification of CaM isotopically enriched for NMR analysis, as well as for the complex of CaM with a peptide derived from the IQ motif sequence of the human cardiac sodium channel NaV1.5. We also describe methods for NMR analysis of titrations of CaM with IQ motif peptides to determine the stoichiometry of the complex and to identify the residues at the binding interface. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Longistatin, an EF-Hand Ca2+-Binding Protein from Vector Tick: Identification, Purification, and Characterization
EF-hand Ca2+-binding motif, a structural component of the EF-hand protein, functions as a calcium sensor and/or buffer in the cytosol of the cell. However, in a few exceptional cases, the EF-hand proteins are secreted from cells and play crucial roles extracellularly. We have identified longistatin, an EF-hand Ca2+-binding protein, from the salivary glands of the tick, Haemaphysalis longicornis. Longistatin possesses an N-terminal sequence of unknown structure and two EF-hand motifs in the C-terminus, which conserve a calmodulin-like canonical structure. Longistatin shows distinct changes in its migration during electropho...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Analysis of Calcium-Induced Conformational Changes in Calcium-Binding Allergens and Quantitative Determination of Their IgE Binding Properties
The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca2+-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Purification and Stable Isotope Labeling of the Calcium- and Integrin-Binding Protein 1 for Structural and Functional NMR Studies
The Calcium- and Integrin-Binding protein 1 (CIB1) has been identified as an important regulatory Ca2+-binding protein that is involved in various cellular functions. Nuclear Magnetic Resonance (NMR) spectroscopy provides a powerful approach to study the structure, dynamics, and interactions of CIB1 and related proteins. Multidimensional NMR spectroscopy combined with various selective isotope labeling strategies has proven to be successful in the structure determination of CIB1. Moreover, the same approach allowed the detection of conformational changes when the protein binds different metal ions, and it facilitated the s...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

X-ray Structural Analysis of S1 Proteins
X-ray crystallography is a potent and meanwhile fast technique to obtain detailed structural information of S100 proteins in their apo or metal ion-loaded state. S100 proteins crystallize in the absence or presence of Ca2+ and Zn2+ and the obtained crystals often diffract to high resolution yielding information on the ion-binding sites, conformation, and target interaction sites of the proteins. Here, I describe a general scheme to isolate and crystallize S100 proteins and the analysis of protein crystals using a modern synchrotron source. (Source: Springer protocols feed by Protein Science)
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Purification and Characterization of the Human Cysteine-Rich S1A3 Protein and Its Pseudo Citrullinated Forms Expressed in Insect Cells
High quantity and quality of recombinant Ca2+-binding proteins are required to study their molecular interactions, self-assembly, posttranslational modifications, and biological activities to elucidate Ca2+-dependent cellular signaling pathways. S100A3 is a unique member of the S100 protein family with the highest cysteine content (10%). This protein, derived from human hair follicles and cuticles, is characterized by an N-terminal acetyl group and irreversible posttranslational citrullination by peptidylarginine deiminase causing its homotetramer assembly. Insect cells, capable of introducing eukaryotic N-terminus and dis...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

New Aspects of Calmodulin–Calmodulin Binding Domains Recognition
Understanding the role of calmodulin (CaM) in calcium signal transduction implies to describe the ­calcium-dependent molecular mechanism of interaction of CaM with the various CaM-binding domains (CBD). In order to fulfill this aim, we have developed a new strategy and the afferent techniques to quantify the interaction of CaM with any CBD as a function of calcium concentration. Excel software has been used to deconvolute the experimental data and to obtain the macroscopic constants characterizing the system. We are illustrating our approach on six different CaM/CBD. This strategy may be used to analyze the interaction...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Super-Resolution Microscopy of the Neuronal Calcium-Binding Proteins Calneuron-1 and Caldendrin
Calcium (Ca2+) signaling in neurons is mediated by plethora of calcium binding proteins with many of them belonging to the Calmodulin family of calcium sensors. Many studies have shown that the subcellular localization of neuronal EF-hand Ca2+-sensors is crucial for their cellular function. To overcome the resolution limit of classical fluorescence and confocal microscopy various imaging techniques have been developed recently that improve the resolution by an order of magnitude in all dimensions. This new microscope techniques make co-localization studies of Ca2+-binding proteins more reliable and help to get insights int...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Probing Ca2+-Binding Capability of Viral Proteins with the EF-Hand Motif by Grafting Approach
Ca2+ is implicated in almost every step of the life cycle of viruses, including virus entry into host cells, virus replication, virion assembly, maturation, and release. However, due to the lack of prediction algorithms and rigorous validation methods, only limited cases of viral Ca2+-binding sites are reported. Here, we introduce a method to predict continuous EF-hand or EF-hand-like motifs in the viral genomes based on their primary sequences. We then introduce a grafting approach, and the use of luminescence resonance energy transfer and Ca2+ competition assay for experimental verification of predicted Ca2+-binding site...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Divers Models of Divalent Cation Interaction to Calcium-Binding Proteins: Techniques and Anthology
Intracellular Ca2+-binding proteins (CaBPs) are sensors of the calcium signal and several of them even shape the signal. Most of them are equipped with at least two EF-hand motifs designed to bind Ca2+. Their affinities are very variable, can display cooperative effects, and can be modulated by physiological Mg2+ concentrations. These binding phenomena are monitored by four major techniques: equilibrium dialysis, fluorimetry with fluorescent Ca2+ indicators, flow dialysis, and isothermal titration calorimetry. In the last quarter of the twentieth century reports on the ion-binding characteristics of several abundant wild-t...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news

Measurement of Intracellular Ca2+ Concentration in Single Cells Using Ratiometric Calcium Dyes
Measurement of intracellular Ca2+ concentration ([Ca2+]i) is useful to study the upstream and downstream events of Ca2+ signaling. Ca2+-binding proteins including EF-hand-containing proteins are important downstream effector molecules after an increase of [Ca2+]i. Conversely, these proteins can also act as key modulators for regulation of [Ca2+]i by sensing the Ca2+ levels in the intracellular organelles and cytoplasm. Here we describe a single-cell Ca2+ imaging technique that was used to measure the intracellular Ca2+ levels to examine the function of Ca2+-binding proteins, STIM1 and Calcium release-activated Calcium chan...
Source: Springer protocols feed by Protein Science - January 1, 2013 Category: Biochemistry Source Type: news