Electrostatics of cysteine residues in proteins: Parameterization and validation of a simple model
ABSTRACT One of the most popular and simple models for the calculation of pK(a) s from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK(a) s. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK(a) s; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK(a) s. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK(a) values (where the calculation should reproduce the pK(a) within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK(a) in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK(a) should be shifted, and validation of force field parameters for cysteine residues. Proteins 2012. © 2012 Wiley Periodicals, Inc.
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ABSTRACT: Either increased protein synthesis or prolonged protein half-life is necessary to support the excessive muscle growth and maintenance of enlarged muscles in myostatin-deficient mice. This issue was addressed by determining in vivo rates of myofibrillar protein synthesis in mice with constitutive myostatin deficiency (Mstn(DeltaE3/DeltaE3)) or normal myostatin expression (Mstn(+/+)) by measuring tracer incorporation after a systemic flooding dose of l-[ring-(2)H(5)]phenylalanine. At 5-6 wk of age, Mstn(DeltaE3/DeltaE3) mice had increased muscle mass (40%), fractional rates of myofibrillar synthesis (14%), and protein synthesis per whole muscle (60%) relative to Mstn(+/+) mice. With maturation, fractional rates of synthesis declined >50% in parallel with decreased DNA and RNA [total, 28S rRNA, and poly(A) RNA] concentrations in muscle. At 6 mo of age, Mstn(DeltaE3/DeltaE3) mice had even greater increases in muscle mass (90%) and myofibrillar synthesis per muscle (85%) relative to Mstn(+/+) mice, but the fractional rate of synthesis was normal. Estimated myofibrillar protein half-life was not affected by myostatin deficiency. Muscle DNA concentrations were reduced in both young and mature Mstn(DeltaE3/DeltaE3) mice, whereas RNA concentrations were normal, so the ratio of RNA to DNA was approximately 30% greater than normal in Mstn(DeltaE3/DeltaE3) mice. Thus the increased protein synthesis and RNA content per muscle in myostatin-deficient mice cannot be explained entirely by an increased number of myonuclei.AJP Endocrinology and Metabolism 03/2006; 290(3):E409-15. DOI:10.1152/ajpendo.00433.2005 · 4.09 Impact Factor
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ABSTRACT: Polyglutamine expansion in certain proteins causes neurodegeneration in inherited disorders such as Huntington disease and X-linked spinobulbar muscular atrophy. Polyglutamine tracts promote protein aggregation in vitro and in vivo with a strict length-dependence that strongly implicates alternative protein folding and/or aggregation as a proximal cause of cellular toxicity and neurodegeneration. We used an intracellular polyglutamine protein aggregation assay based on fluorescence resonance energy transfer (FRET) to identify inhibitors of androgen receptor (AR) aggregation in three libraries of biologically active small molecules: the Annotated Compound Library, the NINDS Custom Collection and a kinase inhibitor collection. In the primary screen 10 compounds reduced AR aggregation. While 10/10 also reduced huntingtin (Htt) exon 1 aggregation, only 2/10 reduced aggregation of pure polyglutamine peptides. In a PC-12 model 9/10 compounds reduced aggregation. Five out of nine compounds tested in an Htt exon 1 assay of neurodegeneration in Drosophila partially rescued the phenotype. Three of the five compounds effective in flies are FDA-approved drugs. These compounds provide new leads for therapeutic development for the polyglutamine diseases based on their efficacy in mammalian cells and a Drosophila model. The high predictive value of the primary screen suggests that the FRET-based screening assay may be useful for further primary and secondary screens for genes or small molecules that inhibit polyglutamine protein aggregation.Human Molecular Genetics 08/2006; 15(13):2114-24. DOI:10.1093/hmg/ddl135 · 6.68 Impact Factor
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ABSTRACT: To characterize and compare electrical myotonia in myotonic dystrophy type 1 (DM1) and type 2 (DM2), 16 patients with genetically confirmed DM1 and 17 patients with DM2 underwent standardized concentric needle electromyography of deltoid, biceps, extensor digitorum communis, first dorsal interosseous, tensor fascia lata (TFL), vastus lateralis (VL), tibialis anterior, and thoracic paraspinal muscles. Eight needle insertions per muscle were made by electromyographers blinded to DM type who recorded the presence and type of myotonia (e.g., classic waxing-waning or less specific waning discharges). Manual muscle testing was performed by a physical therapist. Overall, myotonia was more elicitable in DM1 than DM2; only in VL and TFL was myotonia more elicitable in DM2 than DM1. The major type of myotonia was waxing-waning in DM1, and waning in DM2. Four DM2 (24%), but no DM1 patients had only waning myotonia. In the arms, myotonia was distally predominant in both DM1 and DM2. In the legs, it was distally predominant in DM1, but both proximal and distal in DM2. The severity of myotonia was positively correlated with muscle weakness and with the presence of waxing and waning discharges in DM1, but with neither in DM2. Thus, myotonia is qualitatively and quantitatively different in DM1 than DM2. Except for proximal leg muscles, myotonia is more evocable in DM1 than DM2. It tends to be waxing-waning in DM1 but waning in DM2, thus making electrodiagnosis of DM2 more challenging. Its severity correlates with muscle weakness and the presence of waxing-waning discharges in DM1 but not DM2.Muscle & Nerve 04/2007; 35(4):479-85. DOI:10.1002/mus.20722 · 2.31 Impact Factor