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• Articles by M.J. Jezewska
• Articles by W. Bujalowski

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• Effects of SH1 and SH2 Modifications on Myosin Similarities ...

  Effects of SH1 and SH2 Modifications on Myosin Similarities and Differences Biophysical Journal, Volume 76, Issue 2, 1 February 1999, Pages 1001-1007 Elena A. Bobkova, Andrey A. Bobkov, Dmitrii I. Levitsky and Emil Reisler Abstract The properties of myosin modified at the SH2 group (Cys-697) were studied and compared with the previously reported properties of myosin modified at the SH1 group (Cys-707). 4-[-[(iodoacetoxy)ethyl]- methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) was used for selective modification of the SH2 group on myosin. SH2-labeled heavy meromyosin (SH2-HMM), similar to SH1-labeled HMM (SH1-HMM), did not propel actin filaments in the in vitro motility assays. SH1- and SH2-HMM produced similar amounts of load in the mixtures with unmodified HMM; the sliding speed of actin filaments gradually decreased with an increase in the fraction of either one of the modified HMMs in the mixture. In analogy to SH1-labeled myosin subfragment 1 (SH1-S1), SH2-labeled S1 (SH2-S1) activated regulated actin in the in vitro motility assays. SH2 modification inhibited Mg-ATPase of S1 and its activation by actin. The weak binding of S1 to actin was unaffected whereas the strong binding was weakened by SH2 modification. Overall, our results demonstrate similar behavior of SH1- and SH2-modified myosin heads in the in vitro motility assays despite some differences in their enzymatic properties. The effects of these modifications are ascribed to the location of the SH1-SH2 helix relative to other functional centers of S1. Abstract | Full Text | PDF (129 kb)

• Volatile science? Metabolic engineering of terpenoids in pla...

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• Is SH1-SH2-Cross-Linked Myosin Subfragment 1 a Structural An...

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Abstract

Interactions between the Escherichia coli primary replicative helicase DnaB protein and nucleotide cofactors have been studied using several fluorescent nucleotide analogs and unmodified nucleotides. The thermodynamically rigorous fluorescent titration technique has been used to obtain true binding isotherms, independently of the assumptions of any relationships between the observed quenching of protein fluorescence and the degree of nucleotide binding. Fluorescence titrations using several MANT derivatives of nucleoside diphosphates (MANT-ADP, 3',2'-O-(N-methylantraniloyl)adenosine-5'-diphosphate; MANT-GDP, 3',2'-O(N-methylantraniloyl)guanosine-5'-diphosphate; MANT-CDP, 3',2'-O-(N-methylantraniloyl)cytidine-5'-diphosphate; MANT-UDP, 3',2'-O-(N-methylantraniloyl)uridine-5'-diphosphate) have shown that the DnaB helicase has a preference for purine nucleotides. Binding of all modified nucleotides is characterized by similar negative cooperativity, indicating that negative cooperative interactions are base-independent. Thermodynamic parameters for the interactions of the unmodified nucleotides (ADP, GDP, CDP, and UDP) and inorganic phosphate (P(i)) have been obtained by using the competition titration approach. To analyze multiple ligand binding to a finite circular lattice, for a general case in which each lattice binding site can exist in different multiple states, we developed a matrix method approach to derive analytical expressions for the partition function and the average degree of binding for such cases. Application of the theory to competition titrations has allowed us to extract the intrinsic binding constants and cooperativity parameters for all unmodified ligands. This is the first quantitative estimate of affinities and the mechanisms of binding of different unmodified nucleotides and inorganic phosphate for a hexameric helicase. The intrinsic affinities of all of the studied ATP analogs are lower than the intrinsic affinities of the corresponding ADP analogs. The implications of these results for the mechanism of helicase action are discussed.