Deborah B. Stone

The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study.

The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules.

Chapter 12 Preparation of Deuterated Actin from Dictyostelium discoideum

Publisher Summary This chapter presents the methods that have devised for the production of deuterated Dictyostelium . The method for isolating actin from deuterated Dictyostelium is described as an example for the preparation of deuterated proteins from a eukaryote. The solutions used for extraction and purification can be prepared in H 2 O, as only exchangeable sites will lose deuterium and these are redeuterated by adding D 2 O to the purified protein. The chapter described a procedures using 50–75 g of washed amoebae that are obtained from 5 liters of culture medium. The production of deuterated Dictyostelium represents a major advance in the supply of deuterated eukaryotic macromolecules for structural studies. The procedure outlined is both cost-and time-efficient. D 2 O is the only source of deuterium that must be purchased, and this is recycled. Both E. coli and Dictyostelium readily grow on deuterated medium without need for adaptation and with generation times which are only about twice those on comparable protonated medium. With appropriate modification this procedure should be capable of producing a variety of deuterated macromolecules from Dictyostelium .

The radius of gyration of native and reductively methylated myosin subfragment-1 from neutron scattering.

Reductive methylation of nearly all lysine groups of myosin subfragment-1 (S1) was required for crystallization and solution of its structure at atomic resolution. Possible effects of such methylation on the radius of gyration of chicken skeletal muscle myosin S1 have been investigated by using small-angle neutron scattering. In addition, we have investigated the effect of MgADP.Vi, which is thought to produce an analog of the S1.ADP.Pi state, on the S1 radius of gyration. We find that although methylation of S1, with or without SO42- ion addition, does not significantly alter the structure, addition of ADP plus vanadate does decrease the radius of gyration significantly. The S1 crystal structure predicts a radius of gyration close to that measured here by neutron scattering. These results suggest that the overall shape by crystallography resembles nucleotide-free S1 in solution. In order to estimate the effect of residues missing from the crystal structure, the structure of missing loops was estimated by secondary-structure prediction methods. Calculations using the complete crystal structure show that a simple closure of the nucleotide cleft by a rigid-body torsional rotation of residues (172-180 to 670) around an axis running along the base of the cleft alone does not produce changes as large as seen here and in x-ray scattering results. On the other hand, a rigid body rotation of either the light-chain binding domain (767 to 843 plus light chains) or of a portion of 20-kDa peptide plus this domain (706 to 843 plus light chains) is more readily capable of producing such changes.

Ca ion and the troponin switch.

Muscle contraction as an event manifest by the sliding of myosin filaments along actin filaments was first proposed about fifty years ago by H. Huxley and J. Hanson (Huxley, 2004). This theory built a foundation for muscle research at the molecular level. A decade later the discovery of troponin by Professor S. Ebashi (Ebashi, 1963; Ebashi et al., 1967) highlighted the importance of regulation of muscle contraction and sparked numerous experimental studies of the mysterious protein troponin whose properties are now becoming understood at satisfying resolution.