Protein surface hydration is fundamental to its structure and activity. We report here the direct mapping of global hydration dynamics around a protein in its native and molten globular states, using a tryptophan scan by site-specific mutations. With 16 tryptophan mutants and in 29 different positions and states, we observed two robust, distinct water dynamics in the hydration layer on a few (Ϸ1-8 ps) and tens to hundreds of picoseconds (Ϸ20 -200 ps), representing the initial local relaxation and subsequent collective network restructuring, respectively. Both time scales are strongly correlated with protein's structural and chemical properties. These results reveal the intimate relationship between hydration dynamics and protein fluctuations and such biologically relevant water-protein interactions fluctuate on picosecond time scales.femtosecond dynamics ͉ site-directed mutation ͉ tryptophan scan ͉ water-protein fluctuation W ater motion in the hydration layer is central to protein fluctuation, an essential determinant to its structural stability, dynamics, and function (1-9). Protein surface hydration is a longstanding unresolved problem, but recent extensive studies have merged into a cohesive picture: hydration water molecules are not static but dynamic in nature (1, 2, 10-18). NMR studies (13, 14) have revealed water residence times at protein surfaces within the subnanosecond regime, and molecular dynamics (MD) simulations (15-18) have indicated that water stays in the layer on the time scales from femtoseconds to picoseconds. These processes represent the dynamic exchange of hydration layer water with outside bulk water via thermal fluctuations. Femtosecond-resolved spectroscopic studies of protein solvation (19)(20)(21)(22)(23)(24)(25)(26) recently have shown the dynamics of surface hydration on picosecond time scales with a biphasic distribution. We attributed the first ultrafast solvation to water local relaxation and the second longer-time dynamics to coupled water-protein fluctuations (25,27). To generalize the global heterogeneous hydration dynamics around protein surfaces, correlate the dynamics with protein local structures and chemical identities, and decipher the molecular mechanism of waterprotein fluctuations, we report here our direct mapping of water motions around a globular protein, apomyoglobin (apoMb), in its two states, native and molten globular, using intrinsic tryptophan residue (W) as a local molecular probe to scan the surface by protein engineering.Myoglobin from sperm whale has eight ␣-helices (A-H) with a total of 153 aa (Fig. 1) (28), and all experiments were done with apoMb by removal of the prosthetic heme group. We carefully designed more than 30 mutants and placed tryptophan one at a time along each helix at the protein surface. After we screened all mutant proteins with their structural content, stability, and excited-state lifetime of tryptophan, only 16 mutants are appropriate for mapping global hydration, as shown in Fig. 1. We used a laser wavelength of 290 nm with a pu...

Processivity, the ability of single molecules to move continuously along a track, is a fundamental requirement of cargo-transporting molecular motors. Here, we investigate how cytoplasmic dynein, a homodimeric, microtubule-based motor, achieves processive motion. To do this we developed a versatile method for assembling Saccharomyces cerevisiae dynein heterodimers using complementary DNA oligonucleotides covalently linked to dynein monomers labeled with different organic fluorophores. Using two-color, single-molecule microscopy and high-precision, two-dimensional tracking, we find dynein has a highly variable stepping pattern that is distinct from all other processive cytoskeletal motors, which use “hand-over-hand” mechanisms. Uniquely, dynein stepping is stochastic when its two motor domains are close together. However, coordination emerges as the distance between motor domains increases, implying a tension-based mechanism governs these steps. This plasticity may allow tuning of dynein for the diversity of cellular functions it performs.

The typical fs-resolved fluorescence transients are shown in Figure 2-

SUMMARY The kinesin-8 family of microtubule motors plays a critical role in microtubule length control in cells. These motors have complex effects on microtubule dynamics: they destabilize growing microtubules yet stabilize shrinking microtubules. The budding yeast kinesin-8, Kip3, accumulates on plus ends of growing but not shrinking microtubules. Here we identify an essential role of the tail domain of Kip3 in mediating both its destabilizing and stabilizing activities. The Kip3-tail promotes Kip’s accumulation at the plus ends and facilitates the destabilizing effect of Kip3. However, the Kip3-tail also inhibits microtubule shrinkage and is required for promoting microtubule rescue by Kip3. These effects of the tail domain are likely to be mediated by the tubulin- and microtubule-binding activities that we describe. We propose a concentration-dependent model for the coordination of the destabilizing and stabilizing activities of Kip3 and discuss its relevance to cellular microtubule organization.

Water motion at protein surfaces is fundamental to protein structure, stability, dynamics, and function. By using intrinsic tryptophans as local optical probes, and with femtosecond resolution, it is possible to probe surface-water motions in the hydration layer. Here, we report our studies of local hydration dynamics at the surface of the enzyme Staphylococcus nuclease using site-specific mutations. From these studies of the WT and four related mutants, which change local charge distribution and structure, we are able to ascertain the contribution to solvation by protein side chains as relatively insignificant. We determined the time scales of hydration to be 3-5 ps and 100 -150 ps. The former is the result of local librational͞rotational motions of water near the surface; the latter is a direct measure of surface hydration assisted by fluctuations of the protein. Experimentally, these hydration dynamics of the WT and the four mutants are also consistent with results of the total dynamic Stokes shifts and fluorescence emission maxima and are correlated with their local charge distribution and structure. We discuss the role of protein fluctuation on the time scale of labile hydration and suggest reexamination of recent molecular dynamics simulations.protein hydration ͉ femtosecond dynamics ͉ protein fluctuation ͉ selective mutation F rom the laboratories of the senior authors of this study (A.H.Z. and D.Z.) (1-11), there has been a series of reports regarding the time and length scales of the water layer around protein surfaces. These studies were for proteins subtilisin Carlsberg (2), monellin (3), phospholipase A 2 (5), melittin (9), and human serum albumin (8, 10). A theoretical model was developed to take into account the exchange with bulk water (4, 12), and the dynamics are consistent with molecular dynamics (MD) simulations of residence times (13-16) on time scales from femtoseconds to picoseconds. Earlier NMR studies have reported hydration dynamics (residence times) in the subnanosecond regime (17-20), but, more recently, a claim has been made that water motions at protein surfaces are ultrafast compared with bulk water, only slowing down by a factor of two to three (21,22). This Ͻ10-ps range would imply that the observed long-time hydration dynamics in tens of picoseconds are due to protein side-chain relaxation (22, 23). In our earlier studies (6), we addressed in detail this issue and the reasons for dominance of hydration dynamics. To quantify the contribution of sidechain motions to total solvation on the time scale of hydration, we must carefully alter the local structure while maintaining the same tryptophan site.In this contribution, we report the effect of mutation (four mutants on three site selections and the WT) on hydration of the enzyme Staphylococcus nuclease (SNase). Fig. 1 shows the x-ray structure of the protein, consisting of three ␣-helices and a five-stranded ␤-barrel with a total of 149 amino acids (24). The only single tryptophan residue (W140) has one edge exposed to the surface ...

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SUMMARY The ocular motility disorder “Congenital fibrosis of the extraocular muscles type 1″ (CFEOM1) results from heterozygous mutations altering the motor and 3rd coiled-coil stalk of the anterograde kinesin, KIF21A. We demonstrate that Kif21a knock-in mice harboring the most common human mutation develop CFEOM. The developing axons of the oculomotor nerve’s superior division stall in the proximal nerve; the growth cones enlarge, extend excessive filopodia, and assume random trajectories. Inferior division axons reach the orbit but branch ectopically. We establish a gain-of-function mechanism and find that human motor or stalk mutations attenuate Kif21a autoinhibition, providing in vivo evidence for mammalian kinesin autoregulation. We identify Map1b as a Kif21a interacting protein and report that Map1b−/− mice develop CFEOM. The interaction between Kif21a and Map1b is likely to play a critical role in the pathogenesis of CFEOM1, and highlights a selective vulnerability of the developing oculomotor nerve to perturbations of the axon cytoskeleton.

Computer graphics can not only generate synthetic images and ground truth but it also offers the possibility of constructing virtual worlds in which: (i) an agent can perceive, navigate, and take actions guided by AI algorithms, (ii) properties of the worlds can be modified (e.g., material and reflectance), (iii) physical simulations can be performed, and (iv) algorithms can be learnt and evaluated. But creating realistic virtual worlds is not easy. The game industry, however, has spent a lot of effort creating 3D worlds, which a player can interact with. So researchers can build on these resources to create virtual worlds, provided we can access and modify the internal data structures of the games. To enable this we created an open-source plugin UnrealCV 1 for a popular game engine Unreal Engine 4 (UE4). We show two applications: (i) a proof of concept image dataset, and (ii) linking Caffe with the virtual world to test deep network algorithms.