Ganapathiraman Munussami

Recent applications of bioelectrochemical system for useful resource recovery: retrieval of nutrient and metal from wastewater

Sustainable energy and carbon neutrality has been a key issue in waste/wastewater management. Recovery and recycling of materials such as nutrients and metal are highly anticipated to achieve a zero-energy treatment and pollution control, and subsequently secure sustainability of exhausted resources. The difficulty of resource recovery from waste/wastewater and sediment is mainly due to the immature technology, and dispersed (or less concentrated) target resources in wastewater and natural source that results in technical challenges and poor economics. Bioelectrochemical system (BES) has been widely investigated for electrical energy recovery, intermediate chemical production and recently for useful resource recovery. Certain amounts of energy in wastewater can be used as reducing power for energy production as well as for useful material recovery from wastewater and natural sources using a novel inter-disciplinary biotechnology, BES simultaneously treating wastewater.

A comparative study on the stability and structure of two different green fluorescent proteins in organic co-solvent systems

Green fluorescent protein (GFP) has been used as a reporter marker in a wide range of biological and bioengineering studies. The expanded use of GFP in the field of biosensors, biochips and bio-conjugations requires the stability of GFP in organic co-solvent systems. This prompted us to examine the kinetic stability of two different GFP sequences, n-GFP and s-GFP, showing different folding robustness and thermodynamic stability, under a range of organic co-solvent systems. n-GFP and s-GFP are variants whose biophysical properties are comparable to wild type and super folder GFPs, respectively. The stability of n-GFP and s-GFP in 50% water-miscible organic solvents showed that s-GFP with higher thermodynamic stability exhibited much higher stability against organic solvents than n-GFP, which has lower thermodynamic stability. s-GFP was quite stable even in 90% organic solvents. Circular dichroism analysis confirmed that s-GFP maintained its native structure in organic co-solvent systems, whereas n-GFP showed structural variations under these conditions. Four highly fluctuating loop regions were identified from molecular dynamic simulations under the organic cosolvent conditions. A structural comparison of n-GFP and s-GFP suggested that the improved kinetic stability of s-GFP was due to its larger number of hydrogen bonds and salt-bridges that were present in four loop regions. This study suggests that thermodynamically stable s-GFP can be a good choice for use under harsh organic co-solvent conditions.

Computational screening of potential non-immunoglobulin scaffolds using overlapped conserved residues (OCR)-based fingerprints

Cystatins and lipocalins have attracted considerable interest for their potential applications in non-immunoglobulin protein scaffold engineering. In the present study, their potential homologs were screened computationally from non-redundant protein sequence database based on the overlapped conserved residues (OCR)-fingerprints, which can detect the protein family with low sequence identity, such as cystatins and lipocalins. Two types of OCR-fingerprints for each family were designed and showed very high detection efficiency (>90%). The protein sequence database was scanned by the fingerprints, which yielded the hypothetical sequences for cystatins and lipocalins. The hypothetical sequences were validated further based on their sequence motifs and structural models, which allowed an identification of the potential homologs of cystatins and lipocalins.

In Silico Study on Retinoid-binding Modes in Human RBP and ApoD Lipocalins

Lipocalins are proteins with highly homologous structures but diverse sequences that are potential candidates for scaffold protein engineering with novel ligand-binding functions. Numerous crystal structures of lipocalin-ligand complexes have been identified and used in the study of their binding modes. On the other hand, crystallization studies cannot meet the increasing demand for novel lipocalin-ligand complexes in scaffold engineering, which requires rapid computational analyses of their binding modes in parallel. Human retinol-binding protein (RBP) and apolipoprotein D (apoD) are sequentially very distant proteins, but they show tight binding against retinoids, such as retinol and retinoic acid. In the present study, complexes of the two lipocalins with retinol and retinoic acid were modeled computationally by a molecular docking simulation, and their ligand-binding modes were analyzed at a molecular level. The models identified the crucial residues of lipocalins that interact with the ligands and revealed the similarities and differences in their retinoid-binding modes as well as in the specific interactions of the retinoid species within the same lipocalin. An analysis of the amino acid propensity of the retinoid-binding residues suggested that the evolutionary preference of the residues is restricted to the binding pocket rather than the entire protein. The distribution of charged residues around the terminus of retinoic acid showed a huge difference between RBP and ApoD, which might be a factor for the different binding affinities of lipocalins against retinoic acid. This in silico study is expected to be applied to scaffold protein engineering for novel retinoid-binding lipocalins.

Generation of efficient fingerprint for GFP-like fold and computational identification of potential GFP-like homologs

There is a considerable interest in the detection of GFP-like proteins due to their structural stability and functional usefulness. GFP-like proteins share highly conserved beta-barrel fold with 11 beta-strands. However, their low sequence identity hampers efficient identification of their homologous proteins from database. In this study, an attempt was made to generate a fingerprint for efficient detection of GFP-like proteins. Overlapped conserved residues (OCR)-based approach has been used to design a protein fingerprint based on sequentially and structurally conserved residues in secondary structures to detect homologous proteins very efficiently. Therefore, a fingerprint for GFP-like fold was designed using the OCR approach. However, its specificity was too low to be used for the identification of novel proteins. The conserved residues of loop regions were added and optimized to improve its specificity without losing its high sensitivity. The optimized fingerprint was employed to scan NR database. A total of 20 hypothetical proteins were detected, among which nine were validated as potential GFP-like homologs.