Protein dynamics control proton transfer from bulk solvent to protein interior: a case study with a green fluorescent protein

The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o-nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO(3), and K(2)SO(4), a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.     

Solvent-induced tuning of internal structure in a protein amyloid protofibril

An important goal in studies of protein aggregation is to obtain an understanding of the structural diversity that is characteristic of amyloid fibril and protofibril structures at the molecular level. In this study, what to our knowledge are novel assays based on time-resolved fluorescence anisotropy decay and dynamic quenching measurements of a fluorophore placed at different specific locations in the primary structure of a small protein, barstar, have been used to determine the extent to which the protein sequence participates in the structural core of protofibrils. The fluorescence measurements reveal the structural basis of how modulating solvent polarity results in the tuning of the protofibril conformation from a pair of parallel β-sheets in heat-induced protofibrils to a single parallel β-sheet in trifluorethanol-induced protofibrils. In trifluorethanol-induced protofibrils, the single β-sheet is shown to be built up from in-register β-strands formed by nearly the entire protein sequence, while in heat-induced protofibrils, the pair of β-sheets motif is built up from β-strands formed by only the last two-third of the protein sequence.     

Epidemiological Assessment of Eight Rounds of Mass Drug Administration for Lymphatic Filariasis in India: Implications for Monitoring and Evaluation

Background

Monitoring and evaluation guidelines of the programme to eliminate lymphatic filariasis require impact assessments in at least one sentinel and one spot-check site in each implementation unit (IU). Transmission assessment surveys (TAS) that assess antigenaemia (Ag) in children in IUs that have completed at least five rounds of mass drug administration (MDA) each with >65% coverage and with microfilaria (Mf) levels <1% in the monitored sites form the basis for stopping the MDA. Despite its rigour, this multi-step process is likely to miss sites with transmission potential (‘hotspots’) and its statistical assumptions for sampling and threshold levels for decision-making have not been validated. We addressed these issues in a large-scale epidemiological study in two primary health centres in Thanjavur district, India, endemic for bancroftian filariasis that had undergone eight rounds of MDA.

Methodology/Principal Findings

The prevalence and intensity of Mf (per 60 µl blood) were 0.2% and 0.004 respectively in the survey that covered >70% of 50,363 population. The corresponding values for Ag were 2.3% and 17.3 Ag-units respectively. Ag-prevalence ranged from 0.7 to 0.9%, in children (2–10 years) and 2.7 to 3.0% in adults. Although the Mf-levels in the survey and the sentinel/spot check sites were <1% and Ag-level was <2% in children, we identified 7 “residual” (Mf-prevalence ≥1%, irrespective of Ag-status in children) and 17 “transmission” (at least one Ag-positive child born during the MDA period) hotspots. Antigenaemic persons were clustered both at household and site levels. We identified an Ag-prevalence of ∼1% in children (equivalent to 0.4% community Mf-prevalence) as a possible threshold value for stopping MDA.

Conclusions/Significance

Existence of ‘hotspots’ and spatial clustering of infections in the study area indicate the need for developing good surveillance strategies for detecting ‘hotspots’, adopting evidence-based sampling strategies and evaluation unit size for TAS.     

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.     

Fluorescence spectroscopy for revealing mechanisms in biology: Strengths and pitfalls

This article describes the basic principles of steady-state and time-resolved fluorescence. The formal equivalence of the two methodologies is described first, followed by the extra advantages of time-resolved methods in revealing population heterogeneity in complex systems encountered in biology. Several examples from the author’s work are described in support of the above contention. Finally, several misinterpretations and pitfalls in the interpretation of fluorescence data and their remedy are described.     

Nucleotide sequences of the genes coding for the TEM-like β-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2)

Abstract Two bla _TEM-like genes were characterized that encoded IRT β-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with β-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 β-lactamase resembled the bla _TEM-1B gene, and that for IRT-2 resembled bla _TEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the bla _IRT-1 gene and C to A transversion in the bla _IRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to β-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic p I (p I =5.2) of these IRT enzymes compared to that of TEM-1 (p I =5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this β-lactamase to inhibition by p -chloromercuribenzoate.     

Synthesisin vitro of cholesterol by mitochondria in the shrimpPenaeus aztecus (Ives)

The incorporation of [¹⁴C]-acetate, [¹⁴C]-mevalonate and [¹⁴C]-desmosterol into cholesterol in the muscle mitochondria of the brown shrimpPenaeus aztecus (Ives) is more as compared to that in hepatopancreas. [¹⁴C]-Desmosterol is more efficiently incorporated into cholesterol in comparison with [¹⁴C]-acetate. The muscle mitochondria from males incorporated more [¹⁴C]-mevalonate into cholesterol than those from females, while the converse is true in the hepatopancreatic mitochondria.     

The non-equivalence of binding sites of coenzyme quinone and rotenone in mitochondrial NADH-CoQ reductase

The fluorescent probe erythrosine 5′-iodoacetamide (ER) binds to mitochondrial NADH-CoQ reductase (Complex-I) accompanied by an enhancement of the fluorescence intensity. The binding of the CoQ analogue, 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), decreased the fluorescence intensity of the ER:Complex-I system. The ‘site 1’ inhibitor rotenone did not decrease the fluorescence intensity showing the non-identical nature of the binding sites of DB and rotenone. Also, the reduced form of DB did not decrease the fluorescence intensity. The decrease of the fluorescence intensity by DB was shown to be due to the removal of bound ER by DB. The rapid kinetics of ER binding was studied by temperature-jump relaxation. While DB caused complete elimination of the relaxation process in the ER:Complex-I system, rotenone caused only a decrease in the relaxation rate, suggesting conformational change. The relaxation rate showed a pH dependence with a maximum around pH 7.5.     

Redistribution of nitrogen in an ecosystem due to long-term fertilization and continuous cropping

Summary When a leguminous crop like cowpea was included in a crop rotation of Ganga 5 maize, CO 7 ragi and CO 2 cowpea, the total nitrogen content in the soil was considerably increased even in the unfertilized plots. The leguminous crop fixed atmospheric nitrogen at the rate of 205 kg N/hectare/year. Potassium did not influence the status whereas phosphorus, over a background of N, improved it. Considerable quantity of N fixed was observed to have been redistributed in the soil which depended on the fertilization pattern.