Exploration of fragment-derived modulators of glycoside hydrolases

Previous work at York demonstrated that fragment molecules can increase the activity of the glycoside hydrolase, BtGH84. The initial aim of this project was to use fragment-based discovery methods to identify activators of several enzymes used in cellulose degradation where low activity is one of the limiting steps in the industrial process. This was successful for one enzyme, the fungal glycoside hydrolase, TrBgl2. The characterisation of the mechanism of activation for this enzyme is the main focus of this thesis. A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule activator of TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate binding site, stabilizing the enzyme-substrate complex

Correction to : 1H, 13C, 15N backbone and IVL methyl group resonance assignment of the fungal β-glucosidase from Trichoderma reesei (Biomolecular NMR Assignments, (2020), 10.1007/s12104-020-09959-2)

In the original publication of the article, the name of one of the authors is incorrect. The author's name is Eiso AB, but was modified to A. B. Eiso. The correct name is given in this Correction

1H, 13C, 15N backbone and IVL methyl group resonance assignment of the fungal β-glucosidase from Trichoderma reesei

β-glucosidases have received considerable attention due to their essential role in bioethanol production from lignocellulosic biomass. β-glucosidase can hydrolyse cellobiose in cellulose degradation and its low activity has been considered as one of the main limiting steps in the process. Large-scale conversions of cellulose therefore require high enzyme concentration which increases the cost. β-glucosidases with improved activity and thermostability are therefore of great commercial interest. The fungus Trichoderma reseei expresses thermostable cellulolytic enzymes which have been widely studied as attractive targets for industrial applications. Genetically modified β-glucosidases from Trichoderma reseei have been recently commercialised. We have developed an approach in which screening of low molecular weight molecules (fragments) identifies compounds that increase enzyme activity and are currently characterizing fragment-based activators of TrBgl2. A structural analysis of the 55 kDa apo form of TrBgl2 revealed a classical (α/β)8-TIM barrel fold. In the present study we present a partial assignment of backbone chemical shifts, along with those of the Ile (I)-Val (V)-Leu (L) methyl groups of TrBgl2. These data will be used to characterize the interaction of TrBgl2 with the small molecule activators

Μικρά μόρια - ενεργοποιητές γλυκοσιδικών υδρολασών

Previous work at York demonstrated that fragment molecules can increase the activity of the glycoside hydrolase, BtGH84. The initial aim of this project was to use fragment-based discovery methods to identify activators of several enzymes used in cellulose degradation where low activity is one of the limiting steps in the industrial process. This was successful for oneenzyme, the fungal glycoside hydrolase, TrBgl2. The characterisation of the mechanism of activation for this enzyme is the main focus of this thesis.A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule activator of TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate binding site, stabilizing the enzyme-substrate complex.Προηγούμενη ερευνητική εργασία στο Πανεπιστήμιο του York έδειξε ότι μικρά μόρια μπορούν να αυξήσουν την ενεργότητα της βακτηριακής γλυκοσιδικής υδρολάσης, BtGH84. Ο αρχικός στόχος της παρούσας εργασίας ήταν η χρήση μεθόδων αναζήτησης μικρών μορίων για τον εντοπισμό ενεργοποιητών ενζύμων που χρησιμοποιούνται στην αποικοδόμηση της κυτταρίνης κατά την οποία η χαμηλή ενεργότητα αυτών των ενζύμων αποτελεί ανασταλτικό παράγοντα κατά τη βιομηχανική διαδικασία. Η μέθοδος αναζήτησης μικρών μορίων ήταν επιτυχής στην γλυκοσιδική υδρολάσης ενός μύκητα, TrBgl2. Ο χαρακτηρισμός του μηχανισμού ενεργοποίησης αυτού του ενζύμου είναι το επίκεντρο της παρούσας εργασίας. Μια βιβλιοθήκη 560 εμπορικά διαθέσιμων μικρών μορίων ελέγχθηκε μέσω μιας δοκιμασίας ενεργότητας η οποία εντόπισε ένα μικρό μόριο-ενεργοποιητή του TrBgl2. Ο έλεγχος χημικών αναλόγων του αρχικού μικρού μορίου-ενεργοποιητή και η λεπτομερής κινητική ανάλυση εντόπισε μόρια που συμπεριφέρονταν ως «nonessential» ενεργοποιητές του TrBgl2 και οι οποίοι αυξάνουν έως δύο φορές την μέγιστη ενεργότητα του ενζύμου. Τα μικρά μόρια-ενεργοποιητές δεν επηρέασαν την ενεργότητα της ομόλογης βακτηριακής γλυκοσιδικής υδρολάσης, CcBglA, αποδεικνύοντας την ειδικότητα αυτών των μορίων. Είναι ενδιαφέρον ότι ένα χημικό ανάλογο του αρχικού μικρού μορίου-ενεργοποιητή αναστέλλει τόσο το TrBgl2 όσο και το CcBglA, μέσω του «mixed-model» μηχανισμού. Αν και δεν ήταν δυνατός ο προσδιορισμός της κρυσταλλικής δομής του συμπλέγματος του μικρού μορίου-ενεργοποιητή με το 55 kDa TrBgl2, πειράματα NMR σε διάλυμα έδειξαν μια ειδική θέση δέσμευσής του. Μερική ταυτοποίηση των αμινοξέων του TrBgl2 σε φάσμα NMR προσδιόρισε τα αμινοξέα στη θέση δέσμευσης του μικρού μορίου-ενεργοποιητή, επιτρέποντας τη δημιουργία ενός μοντέλου για την ενεργοποίηση του TrBgl2. Ο ενεργοποιητής προσδένεται στην είσοδο της θέσης δέσμευσης του υποστρώματος, σταθεροποιώντας το σύμπλεγμα ενζύμου-υποστρώματος

Image1_Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA.TIF

The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ70 holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ70. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.</p

Image4_Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA.TIF

The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ70 holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ70. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.</p

Image2_Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA.TIF

The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ70 holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ70. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.</p

Image3_Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA.TIF

The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ70 holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ70. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.</p

DataSheet1_Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA.pdf

The non-coding 6S RNA is a master regulator of the cell cycle in bacteria which binds to the RNA polymerase-σ70 holoenzyme during the stationary phase to inhibit transcription from the primary σ factor. Inhibition is reversed upon outgrowth from the stationary phase by synthesis of small product RNA transcripts (pRNAs). 6S and its complex with a pRNA were structurally characterized using Small Angle X-ray Scattering. The 3D models of 6S and 6S:pRNA complex presented here, demonstrate that the fairly linear and extended structure of 6S undergoes a major conformational change upon binding to pRNA. In particular, 6S:pRNA complex formation is associated with a compaction of the overall 6S size and an expansion of its central domain. Our structural models are consistent with the hypothesis that the resultant particle has a shape and size incompatible with binding to RNA polymerase-σ70. Overall, by use of an optimized in vivo methodological approach, especially useful for structural studies, our study considerably improves our understanding of the structural basis of 6S regulation by offering a mechanistic glimpse of the 6S transcriptional control.</p

The effect of self-talk in learning the volleyball service skill and self-efficacy improvement

In this study the effect of self-talk on learning the volleyball service skill was examined and also the self-efficacy improvement. Participants were 57 female players 13 years old (mean age =12.83, SD=0.97) with two years experience (Μ=1.99, SD=0.67). Prior to the beginning of the program, participants were randomly assigned into two groups: a. the instructional self-talk group (ISTG, n = 28) and b. the control (traditional) group (CG, n = 29). All athletes followed a four-week practice program, aiming at overhand service skill learning and self-efficacy improvement. The program consisted of two practice units (60 min) per week. Participants of ISTG were taught to use the self-talk (for technique) loud before they performed the service drills. The control group received traditional feedback, that is, knowledge of performance and knowledge of results provided by the instructor. Service performance was assessed by videotaped evaluations in five basic elements of skill. There were three measurement periods for field test: pre-, post- and retention tests (one week after post-test). ANOVA repeated measures revealed significant interaction between groups and measures. There was also significant interaction between groups and self-efficacy scores. The results indicated that participants of the ISTG had better scores in the final measurement than the control group, when technique was evaluated and improved also their self-efficacy. In conclusion the Self-talk helps female volleyball athletes to improve performance and learning of overhand service skill and to improve also their self-efficacy. This study adds some useful elements to practitioners and how they used self-talk in the practice