Deciphering ligand specificity of a Clostridium thermocellum family 35 carbohydrate binding module (CtCBM35) for Gluco- and Galacto- Substituted mannans and Its calcium induced stability

Articles in International JournalsThis study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted to 70°C from initial 50°C

Inherent Structural Disorder and Dimerisation of Murine Norovirus NS1-2 Protein

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1–142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein

Proton Magnetic Resonance Spectroscopy Reveals Neuroprotection by Oral Minocycline in a Nonhuman Primate Model of Accelerated NeuroAIDS

Background: Despite the advent of highly active anti-retroviral therapy (HAART), HIV-associated neurocognitive disorders continue to be a significant problem. In efforts to understand and alleviate neurocognitive deficits associated with HIV, we used an accelerated simian immunodeficiency virus (SIV) macaque model of NeuroAIDS to test whether minocycline is neuroprotective against lentiviral-induced neuronal injury. Methodology/Principal Findings: Eleven rhesus macaques were infected with SIV, depleted of CD8+ lymphocytes, and studied until eight weeks post inoculation (wpi). Seven animals received daily minocycline orally beginning at 4 wpi. Neuronal integrity was monitored in vivo by proton magnetic resonance spectroscopy and post-mortem by immunohistochemistry for synaptophysin (SYN), microtubule-associated protein 2 (MAP2), and neuronal counts. Astrogliosis and microglial activation were quantified by measuring glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (IBA-1), respectively. SIV infection followed by CD8+ cell depletion induced a progressive decline in neuronal integrity evidenced by declining N-acetylaspartate/creatine (NAA/Cr), which was arrested with minocycline treatment. The recovery of this ratio was due to increases in NAA, indicating neuronal recovery, and decreases in Cr, likely reflecting downregulation of glial cell activation. SYN, MAP2, and neuronal counts were found to be higher in minocycline-treated animals compared to untreated animals while GFAP and IBA-1 expression were decreased compared to controls. CSF and plasma viral loads were lower in MN-treated animals. Conclusions/Significance: In conclusion, oral minocycline alleviates neuronal damage induced by the AIDS virus

Repression of RNA polymerase by the archaeo-viral regulator ORF145/RIP

Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus two-tailed virus (ATV) forms a high-affinity complex with RNAP by binding inside the DNA-binding channel where it locks the flexible RNAP clamp in one position. This counteracts the formation of transcription pre-initiation complexes in vitro and represses abortive and productive transcription initiation, as well as elongation. Both host and viral promoters are subjected to ORF145 repression. Thus, ORF145 has the properties of a global transcription repressor and its overexpression is toxic for Sulfolobus. On the basis of its properties, we have re-named ORF145 RNAP Inhibitory Protein (RIP)