Maruti Uppalapati

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography.

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF165 to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form ofVEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å2 in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

Anterograde Microtubule Transport Drives Microtubule Bending in LLC-PK1 Epithelial Cells

Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.

Microtubule Alignment and Manipulation Using AC Electrokinetics

The kinesin-microtubule system plays an important role in intracellular transport and is a model system for integrating biomotor-driven transport into microengineered devices. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low-ionic-strength buffers, bundles of microtubules are collected and aligned and the electrical properties of microtubules in solution are measured. The AC electric fields result in electro-osmotic flow, electrothermal flow, and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. By mapping the solution conductivity and AC frequency over which positive dielectrophoresis occurs, the apparent conductivity of taxol-stabilized bovine-brain microtubules in PIPES buffer is measured to be 250 mS m(-1). By maximizing dielectrophoretic forces and minimizing electro-osmotic and electrothermal flow, microtubules are assembled into opposed asters. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications and for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.

Microfabricated capped channels for biomolecular motor-based transport

Kinesins are molecular motors that transport intracellular cargo along microtubules and provide a model system for force generation that can be exploited for biomotor powered nano- and micro-machines. To use this biological system for microscale transport, the most common approach is to reverse the biological geometry and move microtubules along surfaces functionalized with kinesin motors. The microtubules then become potential transport vehicles for sensors and lab-on-a-chip applications. A key requirement for extracting useful work from this system is confinement and control of microtubule movements over kinesin-coated surfaces. The open channel approaches used to date are limited because microtubules that lose contact with the kinesin motors rapidly diffuse away. As a step toward making stand-alone devices incorporating kinesin motors and microtubules, we have developed methods to fabricate capped channels that provide three-dimensional microtubule confinement. We first tested the activity of kinesin motors on a range of surfaces and found that motors were functional on a number of hydrophilic surfaces and nonfunctional on hydrophobic surfaces. In this work, SU-8 photoresist is used to fabricate open channels and a layer of bisbenzocyclobutene (BCB) or dry-film photoresist is used to encapsulate the channels. To allow sample introduction, we fabricate a hierarchical series of microfluidic channels. In this approach, macroscale (/spl sim/250-/spl mu/m) channels in glass or silicon substrates are used to hold fine-gauge stainless steel tubing and allow connection to various fluid sources and intermediate scale (/spl sim/50-/spl mu/m) channels fabricated in thick (/spl sim/50-/spl mu/m) dry-film photoresist are used to connect the macroscale channels to microscale (1-15-/spl mu/m) SU-8 photoresist channels. This paper is the first demonstration of kinesin-based microtubule transport in enclosed microfluidic channels and provides an important step toward packaging these biomolecular motors into functional devices.

KIF14 negatively regulates Rap1a–Radil signaling during breast cancer progression

The kinesin KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules.

Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 surfaces.

Conventional kinesin is routinely adsorbed to hydrophilic surfaces such as SiO(2). Pretreatment of surfaces with casein has become the standard protocol for achieving optimal kinesin activity, but the mechanism by which casein enhances kinesin surface adsorption and function is poorly understood. We used quartz crystal microbalance measurements and microtubule gliding assays to uncover the role that casein plays in enhancing the activity of surface-adsorbed kinesin. On SiO(2) surfaces, casein adsorbs as both a tightly bound monolayer and a reversibly bound second layer that has a dissociation constant of 500 nM and can be desorbed by washing with casein-free buffer. Experiments using truncated kinesins demonstrate that in the presence of soluble casein, kinesin tails bind well to the surface, whereas kinesin head binding is blocked. Removing soluble casein reverses these binding profiles. Surprisingly, reversibly bound casein plays only a moderate role during kinesin adsorption, but it significantly enhances kinesin activity when surface-adsorbed motors are interacting with microtubules. These results point to a model in which a dynamic casein bilayer prevents reversible association of the heads with the surface and enhances association of the kinesin tail with the surface. Understanding protein-surface interactions in this model system should provide a framework for engineering surfaces for functional adsorption of other motor proteins and surface-active enzymes.

A Potent d-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo

Polypeptides composed entirely of d-amino acids and the achiral amino acid glycine (d-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, d-proteins are resistant to degradation by proteases and are anticipated to be nonimmunogenic. Furthermore, d-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed d-protein therapeutic would likely have significant advantages over l-protein drugs. Toward the goal of developing d-protein therapeutics, we previously generated RFX001.D, a d-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (Tm = 33 °C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (Tm > 95 °C), high affinity for VEGF-A (Kd = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the d-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the l-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic d-proteins as alternatives to therapeutic antibodies.

Movement Control of Confined Microtubules

A continuing challenge in nanoscience is the manipulation and assembly of materials at the nanoscale. Kinesin-microtubule biomotors [1] provide a model system for biologically derived nanoscale motion. By using a combination of kinesin functionalization and geometric confinement by channel walls [2], directional movement of microtubules moving over kinesin coated surfaces can be obtained and three-dimensional (3D) confinement allows retention of microtubules for the times and distances required for realistic applications [3]. However, the control of confined microtubules, necessary for most applications, is still poorly understood. We report here on both electric field and surface functionalization approaches to microtubule motion control.

Nano-bio-molecular motors in microfabricated channels

We have investigated hierarchical capped channels that provide 3D microtubule confinement (Huang et al., 2004). We describe here a simplified polymethymethacrylate bonded (PMMA) 3D hierarchical channel process. This process provides shallow motor microchannels (4-6 mum wide and 1.5 mum deep) and deeper structures (~250 mum deep) for fluid input and output and allow simple electrode integration

Channel confined kinesin-microtubule biomolecular nanomotors

Kinesins are molecular motors that move along microtubules, and provide a model system for force generation that can be exploited for kinesin-powered nano- and micro-machines. Microtubules are /spl sim/25 nm diameter cylindrical polymers of the protein tubulin and can be nm to /spl mu/m long. Kinesins bind to microtubules and use the energy of ATP hydrolysis to walk unidirectionally along them at speeds of /spl sim/1 /spl mu/m/s. In this work, we reverse the typical biological system and move microtubules along surfaces functionalized with kinesin motors. The microtubules then become potential transport vehicles for sensors and lab-on-a-chip applications. A key requirement for extracting useful work from this system is confinement and control of the movement of microtubules over kinesin coated surfaces.