Search

This study covers the gender-wise analysis of how behavioural factors and socio-economic factors along with the level of financial literacy influence investment decisions of Indian retail investors. Equally pertinent is to understand that will it have a different influence and bearing on males and females. Multivariate technique partial least squares-structural equation modelling (PLS-SEM) has been applied to develop the model and analyse the results. The study used a structured questionnaire for collecting data from retail investors. The findings of PLS-SEM show that in both genders, behavioural factors, socio-economic factors and financial literacy factors significantly affect investment decisions. However, the findings demonstrate that for women investors, the model is more effective. This study may be useful for prospective fund managers as, in many earlier studies, women are considered to be risk aversive. The results demonstrate that there is a need to target women, and the scenario today is not similar to the pre-existing ones.JEL Classification: G110, G4

Investors differ in their decisions with respect to risk, returns and market analysis. The present study attempts to examine the influence of financial literacy on retail investors' decisions in relation to return, risk and market analysis. The study uses convenience sampling to collect data from the retail investors through stock brokerage managers. Factor analysis has been employed for understanding factors of financial literacy. Financial literacy is composed of Accounting Information; Market Information; Broad Overview; and Technical Knowledge. The factors of Investment decisions are: Return Analytics; Risk Analytics; and Market. The risk and return analytics have stronger impact on investors decision than market analytics. PLS SEM has been employed for examining relation between financial literacy and Investment decision. The results suggest a significant positive relation between financial literacy and investment decision.

This research studies the hotel manager perspective of COVID-19 (the novel coronavirus) on the Indian hotel industry by qualitatively analysing inputs from the human resources department, general management and top management of five-star hotels across India. In doing so, it advances knowledge on the impact of the COVID-19 situation in the domains of human resources (HR), strategy and business operations in the hospitality industry. The study analyses qualitative data collected through online interviews with 17 top-level managers of five-star hotels spread across India. Content analysis is done and the key findings with practical implications have been highlighted. This initial study on the hotel industry in India uncovers how the hoteliers are currently dealing with the pandemic across the country. Findings show that there is a massive negative impact on the Indian hotel industry, in terms of the revenue per available room (RevPAR) and occupancy rates. However, the study also highlights how hoteliers look forward to a phased re-opening of hotels with an increased focus on safety and hygiene after the COVID-19 crisis. The study provides a significant contribution to academic practitioners, hoteliers, and policymakers by examining the future plans in areas of hotels’ HR, strategy and business plans.

Cone beam computer tomography (CBCT) is an advance imaging modality which is very useful for detection of the three dimensional structure or deformity affecting oral and the maxillofacial region and an ideal imaging modality for cystic lesion.1 Nasopalatine cyst is a developmental, epithelial, non-neoplastic cyst that is considered to be the most common non-odontogenic cyst affecting the maxillofacial region.2 These are asymptomatic constituting regular radiological findings, although sometimes patients may report pain due to the compression of structures adjacent to the cyst, it appears as a well-corticated, round or pear shaped radiolucency circumscribed to the maxillary inter incisal region at the midline.2,3 Presenting a case of 29 year male patient who has reported to the department with a chief complain of oozing purulent material from anterior region of palate, which was diagnosed as nasopalatine cyst with the help of CBCT surgical correction was done, with satisfactory healing of lesion in CBCT on 10th month follow up. Keywords: Cone beam computer tomography, Nasopalatine cyst.

Background: Assessment of age through teeth is one of the most reliable and simple method than skeletal remains, to calculate age of an individual. Objectives: The study was carried out with an aim to evaluate reliability of dental age assessment through tooth coronal index (TCI) method. Materials and Methods: The digital panoramic radiographs of 100 subjects of Chhattisgarh aged 20–70 years were selected for the study. The measurements were performed on the JPEG images of selected panoramic radiographs by using Adobe Acrobat 7.0 professional software. The height of the crown, i.e., coronal height, and the height of the coronal pulp cavity, i.e., coronal pulp cavity height, of mandibular second premolars and first molars were measured in millimeter (mm) and then TCI was calculated for each tooth. Actual age of a subject was compared with TCI of tooth and the acquired data were subjected to Pearson's correlation and unpaired t-tests. Results: Negative correlation was observed between the real age and TCI of mandibular first molar (r = −0.092, P = 0.365) and second premolar (r = −0.168, P = 0.096). Statistically significant difference was observed between real age and TCI for mandibular second premolar and first molar (P = 0.000) by unpaired t-test. Conclusion: TCI has the potential to estimate age of an individual on dental radiographs. It is simple, cost effective than histological methods and can be applied to both living and unknown dead.

In the light of rising competition in providing tourism experience amongst Gulf Cooperation Council (GCC) and countries, how efficient Qatar has been in creating tourist satisfaction and tourist motivation in order to create tourist loyalty and promote tourism to global standards. Measuring tourist satisfaction plays an important role in marketing tourism products and services. The purpose of this study is to examine the push and pull factors of tourists visit; their motivation and the activities carry out at the destination. This study explore to understand what comprises tourists are satisfied to transmit their positive experience to third persons as well as repeating their visit to Qatar. The result was drawn from questionnaire survey of 494 international visitors who visited Qatar in 2018. The data were analyzed using matrices which shown the mean scores of expectations, the mean score of experience and the mean of difference between experience and expectation scores. The mean scores of expectation were then plotted against experience in a two dimensional map for positive and negative attributes. The t-test was performed to identify the significant result of each attributes at 1:1000 levels. The findings provide Qatar Tourism Authorities with insights about the level of satisfactions among international tourists and call for better improvements strategies for future tourism development in Qatar.

M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which we discovered two novel classes of inhibitors, 3-cyanopyridones and 1,6-naphthyridin-2-ones, respectively. We describe three cyanopyridone subseries that arose during our hit to lead campaign, along with cocrystal structures of representatives with Mtb TMK. Structure aided optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK. Fragment based lead generation, augmented by crystal structures and the SAR from the cyanopyridones, enabled us to drive the potency of our 1,6-naphthyridin-2-one fragment hit from 500 μM to 200 nM while simultaneously improving the ligand efficiency. Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity against M. tuberculosis. To the best of our knowledge, these compounds are the first reports of non-thymidine-like inhibitors of Mtb TMK.

Dental fractures of the permanent maxillary anterior teeth are relatively frequent accidents during childhood. The Efficient diagnosis and treatment of dental injury are important elements in clinical dentistry. This article describes a case of trauma in permanent right central maxillary incisors with tooth fragments embedded in the lower lip. Thorough clinical examination followed by soft tissue radiographs confirmed the presence of a fractured incisal fragment, which was surgically retrieved under local anesthesia. Direct composite restoration was placed. After finishing and polishing, an esthetic and natural-looking restoration was achieved; this completely satisfied the functional and esthetic expectation of the patient and dental team. How to cite this article: Avinash A, Dubey A, Singh RK, Prasad S. Surgical Removal of Coronal Fragment of Tooth Embedded in Lower Lip and Esthetic Management of Fractured Crown Segment. Int J Clin Pediatr Dent 2014;7(1):65-68.

Differential dual hop relay based system has been introduced to solve the coverage problem. However, fading channel is the weak link of any wireless system. Generalized Gamma (GG) distribution is one of the fading models of wireless communication. However, GG fading channel is not available in any of the software packages. This research paper presents BER analysis of differential dual hop relaying system followed by simulating GG fading channel. Different modulation schemes namely DBPSK and DQPSK have been considered for analyzing the BER of the above system. Furthermore, different channel scenarios between source-relay (SR) and relay-destination (RD) also have been considered for analyzing the above system. Namely, when both SR & RD is slow fading, when SR is fast fading & RD is slow fading and when both SR & RD is fast fading. Numerical analysis also shows the validity of results.

Introduction: Natural herbal remedies have shown promising anti-microbial property and fewer side effects compared to synthetic anti-microbial therapy. Aloe Vera is a medicinal plant used for management of various infections since ancient times as it has anti-inflammatory, anti-microbial, and immuneboosting properties. Aim: The aim of the present study was to determine the antimicrobial and inhibitory activities of various concentration of Aloe Vera Gel (AVG) against oral pathogenic bacteria. Materials and Methods: Subgingival calculus and aspiration of periapical abscess and periodontal abscess was done in 20 patients and the sample transferred to thioglycolate broth, which was incubated in Mutans Sanguis agar, blood agar and cultured in anaerobic gas chamber. The colonies formed were identified further by gram staining methods and biochemical fermentation tests (IMViC). Each isolated colony of identified bacteria were cultured separately in Muller-Hilton broth and incubated at 37°C for 24 hours. Anti-microbial activity of the AVG was tested by the disc diffusion method and minimum inhibitory concentration was determined by broth micro-dilution method. Result: Various staining and biochemical tests confirmed that the sample contained Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), Clostridium bacilli (C. bacilli), Streptcoccus mutans (S. mutans) and Staphlococcus aureus (Staph. aureus). AVG showed anti-bacterial property at 100% and 50% concentration (‘t’ value = 7.504, p-value

The scope of food wastage is wide. This research paper discusses the aspect of avoidable food wastage in hospitality industries in Qatar. The study seeks to find if the hotels can avoid food wastage through considering their food transportation infrastructure or by considering their food portioning while serving the food to the customers. It even seeks to find if the problem of food wastage in hotels can be trimmed if the consumers change their behaviour in terms of buying, preparing and storing food. Three prominently used concept of food waste management were used in hotel industries have been considered. The research methodology uses interpretivist paradigm using SEM to test the six hypotheses with a sample size of 210 managers chosen from 21 five star hotels. Implications have been drawn to benefit the strategic managers to enhance food waste reduction.

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

Putidaredoxin (PdX), the physiological effector of cytochrome P450 cam (P450 cam ), serves to gate electron transfer into oxy-P450 cam during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450 cam turnover reaction. PdX is believed to be difficult to be replaced by an artificial electron donor in the reaction pathway of P450 cam . We demonstrate that the catalytic cycle of wild-type P450 cam can be supported in the presence of an artificial reductant, potassium ferrocyanide. Upon rapid mixing of ferrocyanide ion with P450 cam , we observed an intermediate with spectral features characteristic of compound I. The rate constant for the formation of compound I in the presence of ferrocyanide supported reaction cycle was found to be comparable to the ones observed for H 2 O 2 supported compound I formation in wild-type P450 cam , but was much lower than those observed for classical peroxidases. The results presented in this paper form the first kinetic analysis of this intermediate for an artificial electron-driven P450 cam catalytic pathway in solution.

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.

Na(+) binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na(+) site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na(+) binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na(+). Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na(+) binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na(+) binding and allosteric transduction. X-ray crystal structures of the Na(+)-free (slow) and Na(+)-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-d-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na(+) binding. The slow --fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na(+) to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na(+) binding and the mechanism of thrombin activation by Na(+). The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na(+)-activated enzymes involved in blood coagulation and the immune response.

Objective To assess inter-observer reliability of the identification of episodes of the systemic inflammatory response syndrome (SIRS) in critically ill hospitalized infants and children.

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site. We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes. The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.

This research focuses on the factors influencing student desire to pursue a career in the hospitality industry, with particular reference to hotels. Based on the literature review, seven dimensions have been chosen which have been theoretically linked to the choice of career by students. The dimensions are nature of work, social status, industry-person congeniality, working conditions, pay scale and benefits, promotion opportunities and commitment to the industry. As there is no evidence to empirically prove their relationships, an empirical investigation has been conducted. Second-generation statistical analysis software Structural Equation Modelling has been adopted to test these hypotheses. Two types of analyses were carried out: descriptive statistics and inferential statistics. The descriptive statistics provided the description of the data in terms of Structural Equation Modelling, etc., and the inferential statistics were used to test hypotheses and draw specific suggestions to guide policy makers as to what measures could be taken to build a positive image of the hospitality industry among the future career seekers. Out of the seven hypotheses posited, five have been accepted, based on which suggestions are made.

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate in vivo efficacy against S. aureus in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK.

Secreted proteins play an important part in the pathogenicity of Mycobacterium tuberculosis, and are the primary source of vaccine and diagnostic candidates. A majority of these proteins are exported via the signal peptidase I-dependent pathway, and have a signal peptide that is cleaved off during the secretion process. Sequence similarities within signal peptides have spurred the development of several algorithms for predicting their presence as well as the respective cleavage sites. For proteins exported via this pathway, algorithms exist for eukaryotes, and for Gram-negative and Gram-positive bacteria. However, the unique structure of the mycobacterial membrane raises the question of whether the existing algorithms are suitable for predicting signal peptides within mycobacterial proteins. In this work, we have evaluated the performance of nine signal peptide prediction algorithms on a positive validation set, consisting of 57 proteins with a verified signal peptide and cleavage site, and a negative set, consisting of 61 proteins that have an N-terminal sequence that confirms the annotated translational start site. We found the hidden Markov model of SignalP v3.0 to be the best-performing algorithm for predicting the presence of a signal peptide in mycobacterial proteins. It predicted no false positives or false negatives, and predicted a correct cleavage site for 45 of the 57 proteins in the positive set. Based on these results, we used the hidden Markov model of SignalP v3.0 to analyse the 10 available annotated proteomes of mycobacterial species, including annotations of M. tuberculosis H37Rv from the Wellcome Trust Sanger Institute and the J. Craig Venter Institute (JCVI). When excluding proteins with transmembrane regions among the proteins predicted to harbour a signal peptide, we found between 7.8 and 10.5 % of the proteins in the proteomes to be putative secreted proteins. Interestingly, we observed a consistent difference in the percentage of predicted proteins between the Sanger Institute and JCVI. We have determined the most valuable algorithm for predicting signal peptidase I-processed proteins of M. tuberculosis, and used this algorithm to estimate the number of mycobacterial proteins with the potential to be exported via this pathway.

Background While the genomic annotations of diverse lineages of the Mycobacterium tuberculosis complex are available, divergences between gene prediction methods are still a challenge for unbiased protein dataset generation. M. tuberculosis gene annotation is an example, where the most used datasets from two independent institutions (Sanger Institute and Institute of Genomic Research-TIGR) differ up to 12% in the number of annotated open reading frames, and 46% of the genes contained in both annotations have different start codons. Such differences emphasize the importance of the identification of the sequence of protein products to validate each gene annotation including its sequence coding area. Results With this objective, we submitted a culture filtrate sample from M. tuberculosis to a high-accuracy LTQ-Orbitrap mass spectrometer analysis and applied refined N-terminal prediction to perform comparison of two gene annotations. From a total of 449 proteins identified from the MS data, we validated 35 tryptic peptides that were specific to one of the two datasets, representing 24 different proteins. From those, 5 proteins were only annotated in the Sanger database. In the remaining proteins, the observed differences were due to differences in annotation of transcriptional start sites. Conclusion Our results indicate that, even in a less complex sample likely to represent only 10% of the bacterial proteome, we were still able to detect major differences between different gene annotation approaches. This gives hope that high-throughput proteomics techniques can be used to improve and validate gene annotations, and in particular for verification of high-throughput, automatic gene annotations.

Residue Asp-189 plays an important dual role in thrombin: it defines the primary specificity for Arg side chains and participates indirectly in the coordination of Na(+). The former role is shared by other proteases with trypsin-like specificity, whereas the latter is unique to Na(+)-activated proteases in blood coagulation and the complement system. Replacement of Asp-189 with Ala, Asn, Glu, and Ser drastically reduces the specificity toward substrates carrying Arg or Lys at P1, whereas it has little or no effect toward the hydrolysis of substrates carrying Phe at P1. These findings confirm the important role of Asp-189 in substrate recognition by trypsin-like proteases. The substitutions also affect significantly and unexpectedly the monovalent cation specificity of the enzyme. The Ala and Asn mutations abrogate monovalent cation binding, whereas the Ser and Glu mutations change the monovalent cation preference from Na(+) to the smaller cation Li(+) or to the larger cation Rb(+), respectively. The observation that a single amino acid substitution can alter the monovalent cation specificity of thrombin from Na(+) (Asp-189) to Li(+) (Ser-189) or Rb(+) (Glu-189) is unprecedented in the realm of monovalent cation-activated enzymes.

Monovalent-cation-activated enzymes are abundantly represented in plants and in the animal world. Most of these enzymes are specifically activated by K + , whereas a few of them show preferential activation by Na + . The monovalent cation specificity of these enzymes remains elusive in molecular terms and has not been reengineered by site-directed mutagenesis. Here we demonstrate that thrombin, a Na + -activated allosteric enzyme involved in vertebrate blood clotting, can be converted into a K + -specific enzyme by redesigning a loop that shapes the entrance to the cation-binding site. The conversion, however, does not result into a K + -activated enzyme.

Highly conserved amino acids that form crucial structural elements of the catalytic apparatus can be used to account for the evolutionary history of serine proteases and the cascades into which they are organized. One such evolutionary marker in chymotrypsin-like proteases is Ser(214), located adjacent to the active site and forming part of the primary specificity pocket. Here we report the mutation of Ser(214) in thrombin to Ala, Thr, Cys, Asp, Glu, and Lys. None of the mutants seriously compromises active site catalytic function as measured by the kinetic parameter k(cat). However, the least conservative mutations result in large increases in K(m) because of lower rates of substrate diffusion into the active site. Therefore, the role of Ser(214) is to promote the productive formation of the enzyme-substrate complex. The S214C mutant is catalytically inactive, which suggests that during evolution the TCN-->AGY codon transitions for Ser(214) occurred through Thr intermediates.

The peroxidase activity of carboxymethylated cytochrome c (Cmcytc) has been investigated by spectroscopic and kinetic techniques to examine the effect of carboxymethylation on the peroxidase activity of native cytochrome c (cytc). The optical spectrum suggests that the reaction of Cmcytc with H(2)O(2) proceeds through only one intermediate, compound I. The apparent rate constant (k(app)) for the reaction was found to be 17, 72 and 210 M(-1) s(-1) at pH 7.0, 5.0 and 3.5 respectively. These values are about 60 times larger than those reported for native cytc (0.236 M(-1) s(-1) at pH 7.0), and about five orders of magnitude lower than those for classical peroxidases. Cmcytc was found to catalyse oxidation of organic and inorganic substrates. The second order rate constant for the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) by Cmcytc (205 [H(2)O(2)] s(-1)) is found to be larger than the corresponding value for native cytc (50 [H(2)O(2)] s(-1)) at pH 6.0. The carboxymethylation of cytc ruptures the Fe-S (Met 80) bond and increases the rate of its reaction with H(2)O(2), and its catalytic activity. The specific activity of Cmcytc was measured spectrophotometrically by the reported method using ABTS as substrate, and was found to be 288, 473 and 872 microM min(-1) mg(-1) at pH 7.0, 5.0 and 3.5 respectively. Resonance Raman studies indicated the presence of a bis-histidine coordinated form of Cmcytc at neutral pH, and the existence of a population distribution of different ligation states such as bis-histidine (HH), histidine-water (HW) and five coordinate (5C) forms at lower pH. The relative population of different species in Cmcytc was found to be HH (approximately 100%, approximately 50%, approximately 44%), HW (approximately 0%, approximately 44%, 41%) and 5C (approximately 0%, approximately 6%, 15%) at pH 7.0, 4.7 and 3.1 respectively. We have attempted to correlate the pH dependence of the reaction of Cmcytc with hydrogen peroxide and its peroxidase activity with the haem stereochemical structures observed for Cmcytc. Steady-state and time-resolved tryptophan fluorescence studies on Cmcytc were done to probe the conformational changes around the haem pocket of Cmcytc.

The binding of camphor to cytochrome P450cam has been investigated by steady-state and time-resolved tryptophan fluorescence spectroscopy to obtain information on the substrate access channel. The fluorescence quenching experiments show that some of the tryptophan residues undergo changes in their local environment on camphor binding. The time-resolved fluorescence decay profile gives four lifetime components in the range from 99 ps to 4.5 ns. The shortest lifetime component assigned to W42 lies close to the proposed camphor access channel. The results show that the fluorescence of W42 is greatly affected on binding of camphor, and supports dynamic fluctuations involved in the passage of camphor through the access channel as proposed earlier on the basis of crystallographic, molecular dynamics simulation and site-directed mutagenesis studies.

Binding to helix 12 of the ligand-binding domain of PPARgamma is required for full agonist activity. Previously, the degree of stabilization of the activation function 2 (AF-2) surface was thought to correlate with the degree of agonism and transactivation. To examine this mechanism, we probed structural dynamics of PPARgamma with agonists that induced graded transcriptional responses. Here we present crystal structures and amide H/D exchange (HDX) kinetics for six of these complexes. Amide HDX revealed each ligand induced unique changes to the dynamics of the ligand-binding domain (LBD). Full agonists stabilized helix 12, whereas intermediate and partial agonists did not at all, and rather differentially stabilized other regions of the binding pocket. The gradient of PPARgamma transactivation cannot be accounted for solely through changes to the dynamics of AF-2. Thus, our understanding of allosteric signaling must be extended beyond the idea of a dynamic helix 12 acting as a molecular switch.