Protein Depth Calculation and the Use for Improving Accuracy of Protein Fold Recognition

Protein structure and function are largely specified by the distribution of different atoms and residues relative to the core and surface of the molecule. Relative depths of atoms therefore are key attributions that have been widely used in protein structure modeling and function annotation. However, accurate calculation of depth is time consuming. Here, we developed an algorithm which uses Euclidean distance transform (EDT) to convert the target protein structure into a 3D gray-scale image, where depths of atoms in the protein can be conveniently and precisely derived from the minimum distance of the pixels to the surface of the protein. We tested the proposed EDT-based method on a set of 261 non-redundant protein structures, which shows that the method is 2.6 times faster than the widely used method proposed by Chakravarty and Varadarajan. Depth values by EDT method are highly accurate with a Pearson's correlation coefficient ≈1 compared to the calculations from exhaustive search. To explore the usefulness of the method in protein structure prediction, we add the calculated residue depth to the scoring function of the state of the art, profile?profile alignment based fold-recognition program, which shows an additional 3% improvement in the TM-score of the alignments. The data demonstrate that the EDT-based depth calculation program can be used as an efficient tool to assist protein structure analysis and structure-based function annotation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140343/1/cmb.2013.0071.pd

Dynamic allostery in substrate binding by human thymidylate synthase

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity-a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme

Current state-of-the-art of the research conducted in mapping protein cavities – binding sites of bioactive compounds, peptides or other proteins

Ο σκοπός της διπλωματικής εργασίας είναι η διερεύνηση και αποτύπωση των ερευνητικών μελετών που αφορούν στον χαρακτηρισμό μιας πρωτεϊνικής κοιλότητας – κέντρου πρόσδεσης βιοδραστικών ενώσεων, πεπτιδίων ή άλλων πρωτεϊνών. Στην παρούσα εργασία χρησιμοποιήθηκε η μέθοδος της βιβλιογραφικής επισκόπησης. Παρουσιάζονται τα κυριότερα ευρήματα προηγούμενων ερευνών που σχετίζονται με τη διαδικασία σχεδιασμού φαρμάκων και τον εντοπισμό φαρμακοφόρων με βάση ένα σύνολο προσδετών. Στη συνέχεια συγκρίνονται διαδικασίες επεξεργασίας και ανάλυσης της πρωτεϊνικής κοιλότητας προγενέστερων ερευνών με τη προσέγγιση που προτάθηκε από τους Παπαθανασίου και Φωτόπουλου το 2015. Αναδεικνύονται βασικά πλεονεκτήματα της προσέγγισης αυτής, όπως η εφαρμογή του αλγορίθμου πολυδιάστατη k-means ομαδοποίηση (multidimensional k-means clustering). Η εύρεση βιβλιογραφίας βασίστηκε σε αναζήτηση επιστημονικών άρθρων σε ξενόγλωσσα επιστημονικά περιοδικά, σε κεφάλαια βιβλίων και σε διάφορα άρθρα σε ηλεκτρονικούς ιστότοπους σχετικά με τον σχεδιασμό φαρμάκων και τις κοιλότητες που απαντώνται στις πρωτεΐνες. Στην παρούσα εργασία παρουσιάζονται εν συντομία εργαλεία που εντοπίστηκαν χρησιμοποιώντας λέξεις κλειδιά όπως για παράδειγμα δυναμική πρωτεϊνικής κοιλότητας, καταλυτικό κέντρο ενός ενζύμου, πρόσδεση, πρωτεϊνική θήκη κλπ. Στη συνέχεια συγκροτήθηκε κατάλογος με τα εργαλεία βιοπληροφορικής ανάλυσης που βρέθηκαν και ακολούθησε εκτενής αναφορά επιλεκτικά σε κάποια από αυτά. Κριτήριο επιλογής αυτών των εργαλείων αποτέλεσε η ημερομηνία δημοσίευσής τους, οι αλγόριθμοι και η μεθοδολογία που χρησιμοποιούν. Τα εργαλεία αυτά κατηγοριοποιήθηκαν με βάση τις λέξεις κλειδιά που χρησιμοποιήθηκαν για την εξόρυξη των δεδομένων από την βιβλιογραφία. Τέλος πραγματοποιήθηκε συγκριτική μελέτη αυτών αναδεικνύοντας τα πλεονεκτήματα και εστιάζοντας στην περαιτέρω αξιοποίησή τους.The aim of this thesis was to report on the current state-of-the-art of the research conducted concerning mapping of protein cavities with a potential function role as binding sites of bioactive compounds, peptides or other proteins. A literature review was performed with emphasis on the relevant tools developed during the last decade. In addition, the main research findings regarding drug design and druggable targets based on binding sites are presented. Processes performed in protein cavity detection and analysis, of previous research articles, are compared with the approach described by Anaxagoras Fotopoulos and Athanasios Papathanasiou (2015). The results showed that a competitive advantage of their approach is the multidimensional k-means algorithm for clustering. For the bibliographic review the scientific knowledgebase has been used, which includes international articles and journals, book chapters, as well as online articles regarding drug design and protein cavity. Search keywords such as protein cavity dynamics, catalytic sites of enzymes, protein pocket etc. were used to identify bioinformatics tools with text mining. A catalogue of the most recently developed tools is presented followed by a brief description of selected tools. The selection criteria imposed for preparing the catalogue and the detailed description included the publication date, as well as the algorithms and the methods they use. The tools were then classified according to the search keywords. The findings of this research are discussed, and the algorithms and methods they use are compared, highlighting the advantages of protein cavity detection

Dynamic allostery in substrate binding by human thymidylate synthase

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we demonstrate that hTS binds its nucleotide substrate with ~9-fold entropically-driven positive cooperativity and investigate the mechanism of this binding cooperativity. Characterization of the binding thermodynamics includes ITC and 2D lineshape analysis of NMR titration data. To probe the mechanism of binding cooperativity, we have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity—a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme. We hope that this example will encourage similar studies to reveal how common dynamics and conformational entropy underlie allosteric binding cooperativity.Doctor of Philosoph