Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of similar to 3.5 angstrom in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.

Structure, Spectra and Redox Behaviour of Copper(11) Complexes of Bis( benzimidazoly1)diamine Ligands

The linear quadridentate ligand N,N'-bis(benzimidazoI-2-ylethyl)ethane-l,2-diamine (L') and its 1 - methylbenzimidazole analogue (L2) and homologues form 1 : 1 complexes with Cu(CIO,),; L' also forms complexes of the types CuL'X, where X = NO,, PF,, Br or CI and CuL'(X)Y where X = CI or Br and Y = CIO, or Br. Deep blue CuL1Br,*2H20 crystallizes in the monoclinic space group C2/c with Z = 4, a = 9.91 9(2), b = 16.626(3), c = 14.1 02(3) le\ and p = 94.39(2)". The structure was solved by Patterson and Fourier difference methods and refined by the least-squares technique to R = 0.064 for 2195 independent reflections with / > 1.50(/). The molecule lies on a two-fold axis symmetrically around Cu". The co-ordination around Cu" is found to be square planar with two amino nitrogens and two benzimidazole nitrogens forming the equatorial plane [CU-N 1.983(3) and 2.037(4) A]. The bromides are at longer distances [3.349(1) A] in axial sites. Ligand field and EPR spectra indicate that one bromide or chloride ion is axially co-ordinated to Cu" in [CuL1l2+. This ion exhibits quasi-reversible redox behaviour. Electrochemical studies of the dihalides in methanol have established the presence of [CuL'X,], [CuL'(X)]+ and [CuL'I2+ in equilibrium. In complexes with 565 [CuL4I2+ [L4 = N,Nbis( benzimidazol-2-ylmethyl)ethane-l,2-diamine] and 555 [CuL3] [L3 = N,N'-bis(1 -methylbenzimidazol- 2-ylmethyl)propane-l,3-diamine] chelate rings, Cull does not seem to lie in the N, square plane, as revealed by their low A values and irreversible electrochemical behaviour. The Cu"-Cu' redox potentials in methanol are in the order [CuL1I2+ < [CuL3I2+ < [CuL4I2+; this illustrates that sixmembered chelate rings are suitable to stabilize Cu", when CU-N 0 interactions are favourable.

Seismic Microzonation: Principles,Practices and Experiments

This paper presents an overview of the seismic microzonation and the grade/level based study along with methods used for estimating hazard. The principles of seismic microzonation along with some current practices are discussed. Summary of seismic microzonation experiments carried out in India is presented. A detailed work of seismic microzonation of Bangalore has been presented as a case study. In this case study, a seismotectonic map for microzonation area has been developed covering 350 km radius around Bangalore, India using seismicity and seismotectonic parameters of the region. For seismic microzonation Bangalore Mahanagar Palike (BMP) area of 220 km2 has been selected as the study area. Seismic hazard analysis has been carried out using deterministic as well as probabilistic approaches. Synthetic ground motion at 653 locations, recurrence relation and peak ground acceleration maps at rock level have been generated. A detailed site characterization has been carried out using borehole with standard penetration test (SPT) ―N‖ values and geophysical data. The base map and 3-dimensional sub surface borehole model has been generated for study area using geographical information system (GIS). Multichannel analysis of surface wave (MASW)method has been used to generate one-dimensional shear wave velocity profile at 58 locations and two- dimensional profile at 20 locations. These shear wave velocities are used to estimate equivalent shear wave velocity in the study area at every 5m intervals up to a depth of 30m. Because of wider variation in the rock depth, equivalent shear for the soil overburden thickness alone has been estimated and mapped using ArcGIS 9.2. Based on equivalent shear wave velocity of soil overburden thickness, the study area is classified as ―site class D‖. Site response study has been carried out using geotechnical properties and synthetic ground motions with program SHAKE2000.The soil in the study area is classified as soil with moderate amplification potential. Site response results obtained using standard penetration test (SPT) ―N‖ values and shear wave velocity are compared, it is found that the results based on shear wave velocity is lower than the results based on SPT ―N‖ values. Further, predominant frequency of soil column has been estimated based on ambient noise survey measurements using instruments of L4-3D short period sensors equipped with Reftek 24 bit digital acquisition systems. Predominant frequency obtained from site response study is compared with ambient noise survey. In general, predominant frequencies in the study area vary from 3Hz to 12Hz. Due to flat terrain in the study area, the induced effect of land slide possibility is considered to be remote. However, induced effect of liquefaction hazard has been estimated and mapped. Finally, by integrating the above hazard parameters two hazard index maps have been developed using Analytic Hierarchy Process (AHP) on GIS platform. One map is based on deterministic hazard analysis and other map is based on probabilistic hazard analysis. Finally, a general guideline is proposed by bringing out the advantages and disadvantages of different approaches.

Seismic Microzonation of Bangalore - Preliminary Maps

This paper presents an overview of the seismic microzonation and the grade/level based study along with methods used for estimating hazard. The principles of seismic microzonation along with some current practices are discussed. Summary of seismic microzonation experiments carried out in India is presented. A detailed work of seismic microzonation of Bangalore has been presented as a case study. In this case study, a seismotectonic map for microzonation area has been developed covering 350 km radius around Bangalore, India using seismicity and seismotectonic parameters of the region. For seismic microzonation Bangalore Mahanagar Palike (BMP) area of 220 km2 has been selected as the study area. Seismic hazard analysis has been carried out using deterministic as well as probabilistic approaches. Synthetic ground motion at 653 locations, recurrence relation and peak ground acceleration maps at rock level have been generated. A detailed site characterization has been carried out using borehole with standard penetration test (SPT) ―N‖ values and geophysical data. The base map and 3-dimensional sub surface borehole model has been generated for study area using geographical information system (GIS). Multichannel analysis of surface wave (MASW)method has been used to generate one-dimensional shear wave velocity profile at 58 locations and two- dimensional profile at 20 locations. These shear wave velocities are used to estimate equivalent shear wave velocity in the study area at every 5m intervals up to a depth of 30m. Because of wider variation in the rock depth, equivalent shear for the soil overburden thickness alone has been estimated and mapped using ArcGIS 9.2. Based on equivalent shear wave velocity of soil overburden thickness, the study area is classified as ―site class D‖. Site response study has been carried out using geotechnical properties and synthetic ground motions with program SHAKE2000.The soil in the study area is classified as soil with moderate amplification potential. Site response results obtained using standard penetration test (SPT) ―N‖ values and shear wave velocity are compared, it is found that the results based on shear wave velocity is lower than the results based on SPT ―N‖ values. Further, predominant frequency of soil column has been estimated based on ambient noise survey measurements using instruments of L4-3D short period sensors equipped with Reftek 24 bit digital acquisition systems. Predominant frequency obtained from site response study is compared with ambient noise survey. In general, predominant frequencies in the study area vary from 3Hz to 12Hz. Due to flat terrain in the study area, the induced effect of land slide possibility is considered to be remote. However, induced effect of liquefaction hazard has been estimated and mapped. Finally, by integrating the above hazard parameters two hazard index maps have been developed using Analytic Hierarchy Process (AHP) on GIS platform. One map is based on deterministic hazard analysis and other map is based on probabilistic hazard analysis. Finally, a general guideline is proposed by bringing out the advantages and disadvantages of different approaches.

POLAR NETWORK INDEX AS A MAGNETIC PROXY FOR THE SOLAR CYCLE STUDIES

The Sun has a polar magnetic field which oscillates with the 11 yr sunspot cycle. This polar magnetic field is an important component of the dynamo process which operates in the solar convection zone and produces the sunspot cycle. We have direct systematic measurements of the Sun's polar magnetic field only from about the mid-1970s. There are, however, indirect proxies which give us information about this field at earlier times. The Ca-K spectroheliograms taken at the Kodaikanal Solar Observatory during 1904-2007 have now been digitized with 4k x 4k CCD and have higher resolution (similar to 0.86 arcsec) than the other available historical data sets. From these Ca-K spectroheliograms, we have developed a completely new proxy (polar network index, hereafter PNI) for the Sun's polar magnetic field. We calculate PNI from the digitized images using an automated algorithm and calibrate our measured PNI against the polar field as measured by the Wilcox Solar Observatory for the period 1976-1990. This calibration allows us to estimate the polar fields for the earlier period up to 1904. The dynamo calculations performed with this proxy as input data reproduce reasonably well the Sun's magnetic behavior for the past century.

Synthesis and characterization of monometallic rhenium(I) complexes and their application as selective sensors for copper(II) ions

Novel imine functionalized monometallic rhenium(I) polypyridine complexes (1-4) comprising two phenol moieties attached to 2,20-bipyridine ligands L1-L4 have been synthesized and characterized. These complexes exhibit selective and sensitive detection towards copper(II) ions and this is observed through changes in UV-visible absorption, luminescence and time-resolved spectroscopic techniques. An enormous enhancement is observed in emission intensity, quantum yield and luminescence lifetime with the addition of copper(II) ions, and this can be attributed to the restriction of C=N isomerization in the Re(I) complexes. The strong binding between copper(II) ions and these complexes reveals that the binding constant values are in the range of 1.1 x 10(3)-6.0 x 103 M-1. The absorption spectral behavior of the complexes is supported by DFT calculations.