Controlled activation of protein rotational dynamics using smart hydrogel tethering.

Stimulus-responsive hydrogel materials that stabilize and control protein dynamics have the potential to enable a range of applications that take advantage of the inherent specificity and catalytic efficiencies of proteins. Here we describe the modular construction of a hydrogel using an engineered calmodulin (CaM) within a poly(ethylene glycol) (PEG) matrix that involves the reversible tethering of proteins through an engineered CaM-binding sequence. For these measurements, maltose binding protein (MBP) was isotopically labeled with (13)C and (15)N, permitting dynamic structural measurements using TROSY-HSQC NMR spectroscopy.

Molecular basis of the structural stability of a Top7-based scaffold at extreme pH and temperature conditions.

The development of stable biomolecular scaffolds that can tolerate environmental extremes has considerable potential for industrial and defense-related applications. However, most natural proteins are not sufficiently stable to withstand non-physiological conditions. We have recently engineered the de novo designed Top7 protein to specifically recognize the glycoprotein CD4 by insertion of an eight-residue loop.

Engineering an ultra-stable affinity reagent based on Top7.

Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly stable, protein-based affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail.

Different conformational switches underlie the calmodulin-dependent modulation of calcium pumps and channels.

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM) bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure.

Loss of the calmodulin-dependent inhibition of the RyR1 calcium release channel upon oxidation of methionines in calmodulin.

The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its effect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility determined by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle.

Calcium occupancy of N-terminal sites within calmodulin induces inhibition of the ryanodine receptor calcium release channel.

Calmodulin (CaM) regulates calcium release from intracellular stores in skeletal muscle through its association with the ryanodine receptor (RyR1) calcium release channel, where CaM association enhances channel opening at resting calcium levels and its closing at micromolar calcium levels associated with muscle contraction. A high-affinity CaM-binding sequence (RyRp) has been identified in RyR1, which corresponds to a 30-residue sequence (i.e.

Identification of a denitrase activity against calmodulin in activated macrophages using high-field liquid chromatography--FTICR mass spectrometry.

We have identified a denitrase activity in macrophages that is upregulated following macrophage activation, which is shown by mass spectrometry to recognize nitrotyrosines in the calcium signaling protein calmodulin (CaM). The denitrase activity converts nitrotyrosines to their native tyrosine structure without the formation of any aminotyrosine. Comparable extents of methionine sulfoxide reduction are also observed that are catalyzed by endogenous methionine sulfoxide reductases.

Disruption of interdomain interactions via partial calcium occupancy of calmodulin.

Binding of calcium to CaM exposes clefts in both N- and C-domains to promote their cooperative association with a diverse array of target proteins, functioning to relay the calcium signal regulating cellular metabolism. To clarify relationships between the calcium-dependent activation of individual domains and interdomain structural transitions associated with productive binding to target proteins, we have utilized three engineered CaM mutants that were covalently labeled with N-(1-pyrene) maleimide at introduced cysteines in the C- and N-domains, i.e.

Essential role for Pro21 in phospholamban for optimal inhibition of the Ca-ATPase.

We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP(21), which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro(21) was mutated to an alanine (P21A-PLB(C)); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes.