The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K M (the [ATP] required for half-maximal speed) was 28 ؎ 1 M, and the maximum speed was 904 nm͞s. Pi behaved as a competitive inhibitor with K I ‫؍‬ 9 ؎ 1 mM. ADP behaved approximately as a competitive inhibitor with K I ‫؍‬ 35 ؎ 2 M. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and͞or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P i is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P i before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P i release from the other.crossbridge cycle ͉ motor protein ͉ chemomechanical coupling K inesin is a motor protein that couples the free energy derived from the hydrolysis of ATP into mechanical work used to drive cellular motility. Two properties of kinesin are essential for its function. First, kinesin undergoes directed motion. It moves toward the plus-or fast-growing end of a microtubule as it transports membrane-bounded organelles toward the periphery of neurons and other cells (1) where the plus ends of microtubules are usually located (2). And second, kinesin is processive. An individual kinesin molecule can move up to several microns along a microtubule without dissociating (3). Processivity ensures that even a small vesicle with just one or two motors on its surface will spend a large fraction of its time attached to and moving along a microtubule (as opposed to diffusing in the cytoplasm).The directed and processive motion of kinesin is tightly coupled to the hydrolysis of ATP. High-precision tracking of kinesin-coated beads reveals that kinesin takes 8-nm steps (4) from one tubulin dimer to the next along a path that is parallel to the axis of the microtubule (5). Direct measurement of the ATPase rate and its correlation to the speed of movement indicates that in standard motility assays where the load is low, kinesin hydrolyses only one ATP per 8-nm step (6). A stoichiometry of 1 step per ATP implies that each cycle of ATP hydrolysis [the binding of ATP to kinesin's nucleotide-binding pocket, its hydrolysi...

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solidstate NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constantcomposition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13 C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ؎ 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete ␣-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an ␣-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

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