Computational Design of Novel Proteins for Affinity Reagents

Computational protein design is an emerging technology that can pave a way to redesign and create novel proteins which have new functions as affinity reagents and protein therapeutics. Two approaches have been described to create an affinity reagent for activated form of p21-activated kinase 1 (PAK1). A good affinity reagent can be developed into a biosensor for visualizing the activated form of PAK1 inside cells. In the first approach, truncated auto-inhibitory domain (trunc-AID) from PAK1, which is unfolded but binds the activated conformation of PAK1 with an affinity of 4 [mu]M, was redesigned for improvement in structure and function. Using a computational approach implemented within Rosetta molecular modeling program, a 20-residue helix at the N-terminus was created in the N-terminus of trunc-AID that had the PAK1-binding region conserved. The design, AlmostHelix showed gain in structure and solubility but no improvement in binding affinity. Another design, PAcKer, where a 16-residue helix was created at the C-terminus of the trunc-AID and was inserted in a fluorescent protein (CFP) bound the active conformation of PAK1 with an affinity of ~400 nM. CFP-PAcKer did not show any binding to the 'closed' form of PAK1 making it suitable for a biosensor that can detect only the activated form of PAK1 inside cells. A de novo interface design approach was used to create a binding interface on a segment from hyperplastic discs protein ('scaffold') to bind the active conformation of PAK1 ('target'). Using a newly developed protocol DDMI, that is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side chain torsion angles to design low energy interfaces between the 'scaffold' and 'target', a binder called Spider Roll was designed. Spider Roll bound to PAK1 with a modest affinity of 100 [mu]M. Mutagenesis studies confirmed that the binding interface was consistent with the design model. NMR studies were also consistent with the binding model. Additionally, Spider Roll did not bind the 'closed' PAK1. De novo designed binders that can distinguish between two states of proteins have potential use as biosensors. As a future direction for de novo protein interface design, an attempt was made to create a metal-mediated interaction between a 'scaffold' protein and Ubiquitin as a 'target'. Metal-mediated interactions have advantage over other non-covalent interactions due to strong coordination bond. Experimental testing of multiple designs showed a design (called Spelter) bound with low nanomolar affinity to the metal ion, but any metal-mediated interaction between Spelter and Ubiquitin was not established

Metallurgical investigation of cracked Al–5.5Zn–2.5Mg–1.5Cu aluminium alloy valve

AbstractThe high strength aluminium alloy Al–5.5Zn–2.5Mg–1.5Cu (AA7075) is being widely used in realisation of aerospace components. A component ‘fill and vent valve’ used in liquid propulsion system was fabricated from AA 7075 forgings in T7352 temper condition, and subsequently undergone various functional tests, four years back. Recently, during dye penetrant test after proof pressure test at 525bar, a valve indicated presence of a crack. Detailed metallurgical investigation indicated that failure was caused by stress corrosion cracking

Nanomedicines for the management of diabetic nephropathy: present progress and prospects

Diabetic nephropathy (DN) is a serious microvascular consequence of diabetes mellitus (DM), posing an encumbrance to public health worldwide. Control over the onset and progress of DN depend heavily on early detection and effective treatment. DN is a major contributor to end-stage renal disease, and a complete cure is yet to be achieved with currently available options. Though some therapeutic molecules have exhibited promise in treating DN complications, their poor solubility profile, low bioavailability, poor permeation, high therapeutic dose and associated toxicity, and low patient compliance apprehend their clinical usefulness. Recent research has indicated nano-systems as potential theranostic platforms displaying futuristic promise in the diagnosis and treatment of DN. Early and accurate diagnosis, site-specific delivery and retention by virtue of ligand conjugation, and improved pharmacokinetic profile are amongst the major advantages of nano-platforms, defining their superiority. Thus, the emergence of nanoparticles has offered fresh approaches to the possible diagnostic and therapeutic strategies regarding DN. The present review corroborates an updated overview of different types of nanocarriers regarding potential approaches for the diagnosis and therapy of DN

Mapping ecologically sensitive, significant and salient areas of Western Ghats: proposed protocols and methodology

The Western Ghats Ecology Expert Panel (WGEEP) of the Ministry of Environment and Forests, Government of India (GOI) has been asked to identify ecologically sensitive areas (ESAs) along the Western Ghats, and to suggest how to manage them. The concept of ESAs has been extensively discussed in the literature. Several ESAs have been set up in India over the last 22 years under the Environment Protection Act, 1986, and a GOI committee under the chairmanship of Pranob Sen has proposed certain criteria for identification of ESAs. However, WGEEP noted that we still lack a global consensus either on the criteria to define ESAs or on a workable methodology to identify them. Furthermore, there are no clear guidelines on the management regime that should prevail in ESAs, and the Pranob Sen Committee has not addressed this issue at all. Hence, WGEEP decided to undertake an exercise of defining ESAs and developing a workable methodology to assign levels of ecological significance/sensitivity as a first step towards putting ESAs on the map of the Western Ghats. This article provides a report on the outcome of a series of discussions and consultations held by WGEEP to build a consensus on defining and mapping ESAs. It hopes to provoke discussion and feedback from a wider section of experts, with the aim of finalizing a generic methodology for mapping ESAs in other ecologically sign ificant, biodiversity-rich areas within and outside the country. We hope to shortly prepare a companion paper that will address the equally vital management issues

Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments

Redesign of the PAK1 Autoinhibitory Domain for Enhanced Stability and Affinity in Biosensor Applications

The inhibitory switch (IS) domain of p21-activated kinase 1 (PAK1) stabilizes full-length PAK1 in an inactive conformation by binding to the PAK1 kinase domain. Competitive binding of small GTPases to the IS domain disrupts the autoinhibitory interactions and exposes the IS domain binding site on the surface of the kinase domain. To build an affinity reagent that selectively binds the activated state of PAK1, we used molecular modeling to re-engineer the isolated IS domain so that it was soluble and stable, did not bind to GTPases and bound more tightly to the PAK1 kinase domain. Three design strategies were tested: in the first and second case, extension and redesign of the N-terminus were used to expand the hydrophobic core of the domain and in the third case the termini were redesigned to be adjacent in space so that that the domain could be stabilized by insertion into a loop in a host cyan fluorescent protein (CFP). The best-performing design, called CFP-PAcKer, was based on the third strategy and bound the kinase domain of PAK1 with an affinity of 400 nM. CFP-PAcKer binds more tightly to a full-length variant of PAK1 that is stabilized in the ‘open’ state (Kd = 3.3 µM) than to full-length PAK1 in the ‘closed’ state (undetectable affinity), and binding can be monitored with fluorescence by placing an environmentally sensitive fluorescence dye on CFP-PAcKer adjacent to the binding site

Computational Design of a PAK1 Binding Protein

We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side chain torsion angles to design low energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 μM. The tightest binding design, named Spider Roll, bound with an affinity of 100 μM. NMR –based structure prediction of Spider Roll based on backbone and 13Cβ chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full length PAK1 in its activated state, but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding `on-rate' constant (<< 105 M−1 s−1). The ability to rationally design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful as therapeutics or molecular probes

Single lesion multibacillary leprosy, a treatment enigma: a case report

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Male Use of Female Sex Work in India: A Nationally Representative Behavioural Survey

Heterosexual transmission of HIV in India is driven by the male use of female sex workers (FSW), but few studies have examined the factors associated with using FSW. This nationally representative study examined the prevalence and correlates of FSW use among 31,040 men aged 15–49 years in India in 2006. Nationally, about 4% of men used FSW in the previous year, representing about 8.5 million FSW clients. Unmarried men were far more likely than married men to use FSW overall (PR = 8.0), but less likely than married men to use FSW among those reporting at least one non-regular partner (PR = 0.8). More than half of all FSW clients were married. FSW use was higher among men in the high-HIV states than in the low-HIV states (PR = 2.7), and half of all FSW clients lived in the high-HIV states. The risk of FSW use rose sharply with increasing number of non-regular partners in the past year. Given the large number of men using FSW, interventions for the much smaller number of FSW remains the most efficient strategy for curbing heterosexual HIV transmission in India