Retention of Conformational Entropy upon Calmodulin Binding to Target Peptides Is Driven by Transient Salt Bridges

AbstractCalmodulin (CaM) is a highly flexible calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. To identify how binding affects CaM motions, and its relationship to conformational entropy and target peptide sequence, we have employed fully atomistic, explicit solvent molecular dynamics simulations of unbound CaM and CaM bound to five different target peptides. The calculated CaM conformational binding entropies correlate with experimentally derived conformational entropies with a correlation coefficient R2 of 0.95. Selected side-chain interactions with target peptides restrain interhelical loop motions, acting to tune the conformational entropy of the bound complex via widely distributed CaM motions. In the complex with the most conformational entropy retention (CaM in complex with the neuronal nitric oxide synthase binding sequence), Lys-148 at the C-terminus of CaM forms transient salt bridges alternating between Glu side chains in the N-domain, the central linker, and the binding target. Additional analyses of CaM structures, fluctuations, and CaM-target interactions illuminate the interplay between electrostatic, side chain, and backbone properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy, and suggest a need to consider conformational dynamics in optimizing binding affinities

Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase

AbstractMethionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D-sulfoxide diastereomer of CaM-bound L-MetSO (L-Met-D-SO). After exhaustive reduction by pMSR, the ratio of L-Met-D-SO to L-Met-L-SO decreased to about 1:25 for hydrogen peroxide-oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR

A Top-Down LC-FTICR MS-Based Strategy for Characterizing Oxidized Calmodulin in Activated Macrophages

A liquid chromatography-mass spectrometry (LC-MS)-based approach for characterizing the degree of nitration and oxidation of intact calmodulin (CaM) has been used to resolve ∼250 CaM oxiforms using only 500 ng of protein. The analysis was based on high-resolution data of the intact CaM isoforms obtained by Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) coupled with an on-line reversed-phase LC separation. Tentative identifications of post-translational modifications (PTMs), such as oxidation or nitration, have been assigned by matching observed protein mass to a database containing all theoretically predicted oxidation products of CaM and verified through a combination of tryptic peptide information (generated from bottom-up analyses) and on-line collisionally induced dissociation (CID) tandem mass spectrometry (MS/MS) at the intact protein level. The reduction in abundance and diversity of oxidatively modified CaM (i.e., nitrated tyrosines and oxidized methionines) induced by macrophage activation has been explored and semiquantified for different oxidation degrees (i.e., no oxidation, moderate, and high oxidation). This work demonstrates the power of the top-down approach to identify and quantify hundreds of combinations of PTMs for single protein target such as CaM and implicate competing repair and peptidase activities to modulate cellular metabolism in response to oxidative stress

Loss of Conformational Stability in Calmodulin upon Methionine Oxidation

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent α-helical content at 28°C, and only a small (0.3 ± 0.1 kcal mol−1) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM

Proteomic Analysis of Salmonella enterica Serovar Typhimurium Isolated from RAW 264.7 Macrophages

n/

GPS-CCD: A Novel Computational Program for the Prediction of Calpain Cleavage Sites

As one of the most essential post-translational modifications (PTMs) of proteins, proteolysis, especially calpain-mediated cleavage, plays an important role in many biological processes, including cell death/apoptosis, cytoskeletal remodeling, and the cell cycle. Experimental identification of calpain targets with bona fide cleavage sites is fundamental for dissecting the molecular mechanisms and biological roles of calpain cleavage. In contrast to time-consuming and labor-intensive experimental approaches, computational prediction of calpain cleavage sites might more cheaply and readily provide useful information for further experimental investigation. In this work, we constructed a novel software package of GPS-CCD (Calpain Cleavage Detector) for the prediction of calpain cleavage sites, with an accuracy of 89.98%, sensitivity of 60.87% and specificity of 90.07%. With this software, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. In this regard, GPS-CCD 1.0 is considered to be a useful tool for experimentalists. The online service and local packages of GPS-CCD 1.0 were implemented in JAVA and are freely available at: http://ccd.biocuckoo.org/

De novo mutations in GRIN1 cause extensive bilateral polymicrogyria

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria

Differences in microbial metabolites in urine headspace of subjects with Immune Thrombocytopenia (ITP) detected by volatile organic compound (VOC) analysis and metabolomics

ITP is an organ-specific autoimmune disorder characterised by a low platelet count whose cause is uncertain. A possible factor is food intolerance, although much of the information linking this with ITP is anecdotal. The role of food intolerance in ITP was studied by replacing a normal diet with an elemental diet (E028), but this did not increase platelet counts. Clear differences, however, were apparent between the volatile organic compounds (VOCs) in the urine headspace of patients with ITP and those present in healthy volunteers, which leads to speculation that abnormal metabolic activity of the intestinal microbiome may be a factor causing ITP. However, further work is needed to confirm this. There were also differences between the VOCs of patients on a normal diet and those on the elemental diet, and in this case, the VOCs involved are very likely to be of bacterial origin, as their production is affected by dietary manipulation. Many of these VOCs are known to be toxic

Technologies of contraception and abortion

Soon to turn 60, the oral contraceptive pill still dominates histories of technology in the ‘sexual revolution’ and after. ‘The pill’ was revolutionary for many, though by no means all, women in the west, but there have always been alternatives, and looking globally yields a different picture. The condom, intrauterine device (IUD), surgical sterilization (male and female) and abortion were all transformed in the twentieth century, some more than once. Today, female sterilization (tubal ligation) and IUDs are the world's most commonly used technologies of contraception. The pill is in third place, followed closely by the condom. Long-acting hormonal injections are most frequently used in parts of Africa, male sterilization by vasectomy is unusually prevalent in Britain, and about one in five pregnancies worldwide ends in induced abortion. Though contraceptive use has generally increased in recent decades, the disparity between rich and poor countries is striking: the former tend to use condoms and pills, the latter sterilization and IUDs. Contraception, a term dating from the late nineteenth century and since then often conflated with abortion, has existed in many forms, and techniques have changed and proliferated over time. Diverse local cultures have embraced new technologies while maintaining older practices. Focusing on Britain and the United States, with excursions to India, China and France, this chapter shows how the patterns observed today were established and stabilized, often despite persistent criticism and reform efforts. By examining past innovation, and the distribution and use of a variety of tools and techniques, it reconsiders some widely held assumptions about what counts as revolutionary and for whom. Analytically, it takes up and reflects on one of the main issues raised by feminists and social historians: the agency of users as patients and consumers faced with choice and coercion. By examining practices of contraception alongside those of abortion, it revisits the knotty question of technology in the sexual revolution and the related themes of medical, legal, religious and political forms of control