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Indian Institute of Science

1. Hatti, Kaushik S. Structure Determination of Proteins of Unknown Origin by a Marathon MR Protocol and Investigations on Parameters Important for Molecular Replacement Structure Solution.

Degree: 2016, Indian Institute of Science

Occasionally, crystallisation of proteins works in mysterious ways! One might obtain crystals of a protein of unknown identity in place of the protein for which crystallisation experiments were performed. If the investigator is not aware of such possibilities, valuable time and resources might be lost in attempting to determine the structure of such proteins. Instances of non-target protein getting crystallised may not come to light at all or may be realised only when attempts to determine the structure completely fail by conventional procedures after collecting and processing the diffraction data. Usually, it is not possible to reproduce the crystals of the same protein as their occurrence is serendipitous. Such rare instances of crystallisation are probably caused by fluctuating environmental or crystallisation conditions and are not reproducible. It could also be due to contaminating microbes, which is more likely when the experimentalist is not well experienced. Therefore, experimental phasing of the data collected on serendipitously obtained crystals could be a challenging task. With the rapid increase in the number of structures deposited in the protein data bank (PDB), molecular replacement has become the method of choice for structure determination in macromolecular X-ray crystallography. This is due to the fact that it is possible to select a suitable phasing model for most target proteins based on their sequence information. However, if the identity of the target protein itself is uncertain, all attempts of structure determination using phasing models selected on the basis of target protein sequence-dependent search would fail. Sequence-independent ab initio phasing techniques such as ARCIMBOLDO (Meindl et al., 2012), which has recently become available, could provide leads only if the non-target protein is an all-α-protein and the associated diffraction data extends to a resolution better than 2 Å. Even then, the success rate with this technique is low. Hence, it becomes important to employ a sequence-independent method of structure determination for such mysteriously obtained crystals. This thesis reports crystal structures of proteins which are serendipitously crystallised using a large-scale application of Molecular Replacement (MR) technique (referred in this thesis as MarathonMR). This thesis also presents an evaluation of molecular replacement strategies for structure determination. The thesis begins with an overview of crystallographic methods of structure determination with an emphasis on the method of molecular replacement (Chapter 1). The most prominent of the results obtained in the course of these investigations pertains to a crystal obtained during routine crystallisation of a viral protein mutant in the year 2011. The cell parameters were different from cell constants of crystals obtained with other known viral protein mutants crystallised earlier in the same laboratory. Unfortunately, this crystal could not be reproduced in the same form in subsequent crystallisation trials. All attempts… Advisors/Committee Members: Srinivasan, N, Murthy, M R N.

Subjects/Keywords: Proteins Structure Determination; Crystal Systems; Protein Crystallisation; Molecular Replacement (MR); Crystallisation; Stenotrophomonas maltophilia Phosphate Binding Protein; Marathon MR Procedure; Crystal Structures of Proteins; Crystallisation of Proteins; Molecular Biophysics

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Hatti, K. S. (2016). Structure Determination of Proteins of Unknown Origin by a Marathon MR Protocol and Investigations on Parameters Important for Molecular Replacement Structure Solution. (Thesis). Indian Institute of Science. Retrieved from http://hdl.handle.net/2005/3148

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Hatti, Kaushik S. “Structure Determination of Proteins of Unknown Origin by a Marathon MR Protocol and Investigations on Parameters Important for Molecular Replacement Structure Solution.” 2016. Thesis, Indian Institute of Science. Accessed July 19, 2019. http://hdl.handle.net/2005/3148.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Hatti, Kaushik S. “Structure Determination of Proteins of Unknown Origin by a Marathon MR Protocol and Investigations on Parameters Important for Molecular Replacement Structure Solution.” 2016. Web. 19 Jul 2019.

Vancouver:

Hatti KS. Structure Determination of Proteins of Unknown Origin by a Marathon MR Protocol and Investigations on Parameters Important for Molecular Replacement Structure Solution. [Internet] [Thesis]. Indian Institute of Science; 2016. [cited 2019 Jul 19]. Available from: http://hdl.handle.net/2005/3148.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Hatti KS. Structure Determination of Proteins of Unknown Origin by a Marathon MR Protocol and Investigations on Parameters Important for Molecular Replacement Structure Solution. [Thesis]. Indian Institute of Science; 2016. Available from: http://hdl.handle.net/2005/3148

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

2. ΓΕΛΑΔΟΠΟΥΛΟΣ, ΤΑΞΙΑΡΧΗΣ. ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΣΕ ΕΓΚΕΦΑΛΟ ΘΗΛΑΣΤΙΚΩΝ.

Degree: 1996, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών (ΕΚΠΑ); National and Kapodistrian University of Athens

Ο ΜΗΧΑΝΙΣΜΟΣ ΤΗΣ ΑΠΟΦΩΣΦΟΡΥΛΙΩΣΗΣ ΤΩΝ ΠΟΛΥΦΩΣΦΟΡΥΛΙΩΜΕΝΩΝ ΠΡΩΤΕΙΝΩΝ ΣΤΟΝ ΕΓΚΕΦΑΛΟ ΤΩΝ ΘΗΛΑΣΤΙΚΩΝ ΔΕΝ ΕΧΕΙ ΚΑΤΑΝΟΗΘΕΙ ΕΠΑΡΚΩΣ. ΧΡΗΣΙΜΟΠΟΙΩΝΤΑΣ ΣΑΝ ΥΠΟΣΤΡΩΜΑ-ΜΟΝΤΕΛΟ ΤΗΝ ΕΝΔΟΓΕΝΩΣ ΠΟΛΥΦΩΣΦΟΡΥΛΙΩΜΕΝΗ ΠΡΩΤΕΙΝΗ ΦΩΣΒΙΤΙΝΗ, ΕΠΙΧΕΙΡΗΣΑΜΕ ΝΑ ΑΠΟΜΟΝΩΣΟΥΜΕ ΚΑΙ ΝΑ ΧΑΡΑΚΤΗΡΙΣΟΥΜΕ ΤΙΣ ΦΩΣΦΑΤΑΣΕΣ ΑΠΟ ΕΓΚΕΦΑΛΟ ΑΡΟΥΡΑΙΟΥ ΠΟΥ ΑΠΟΦΩΣΦΟΡΥΛΙΩΝΟΥΝ ΚΑΤΑ ΠΡΟΤΙΜΗΣΗ ΠΟΛΥΦΩΣΦΟΡΥΛΙΩΜΕΝΕΣ ΠΡΩΤΕΙΝΕΣ. ΑΝΑΠΤΥΞΑΜΕ ΜΙΑ ΑΠΛΗ ΚΑΙ ΕΥΑΙΣΘΗΤΗ ΧΡΩΜΑΤΟΜΕΤΡΙΚΗ ΜΕΘΟΔΟ ΠΡΟΣΔΙΟΡΙΣΜΟΥ ΔΡΑΣΤΙΚΟΤΗΤΑΣ ΦΩΣΦΑΤΑΣΩΝ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΜΕ ΒΑΣΗ ΤΟ ΠΡΑΣΙΝΟ ΤΟΥ ΜΑΛΑΧΙΤΗ. ΔΥΟ ΔΡΑΣΤΙΚΟΤΗΤΕΣ ΦΩΣΦΑΤΑΣΗΣ ΤΗΣ ΦΩΣΒΙΤΙΝΗΣ, ΠΟΥ ΑΠΟΚΑΛΟΥΝΤΑΙ ΦΩΣΦΑΤΑΣΗ ΤΗΣ ΦΩΣΒΙΤΙΝΗΣ 1 ΚΑΙ 2 (PVP1 ΚΑΙ PVP2), ΑΠΟΜΟΝΩΘΗΚΑΝ ΑΠΟ ΕΓΚΕΦΑΛΟ ΑΡΟΥΡΑΙΟΥ ΚΑΙ ΧΑΡΑΚΤΗΡΙΣΤΗΚΑΝ. ΚΑΙ ΟΙ ΔΥΟ ΦΩΣΦΑΤΑΣΕΣ ΑΠΟΦΩΣΦΟΡΥΛΙΩΝΟΥΝ ΕΠΙΣΗΣ (I) ΤΗ ΦΩΣΦΟΡΥΛΑΣΗ A, (II) ΤΙΣ Α ΚΑΙ Β ΥΠΟΜΟΝΑΔΕΣ ΤΗΣ ΚΙΝΑΣΗΣ ΤΗΣ ΦΩΣΦΟΡΥΛΑΣΗΣ, (III) ΤΗΝ ΠΡΩΤΕΙΝΗ ΠΟΥ ΣΥΝΔΕΕΤΑΙ ΜΕ ΜΙΚΡΟΣΩΛΗΝΙΣΚΟΥΣ Τ, (IV) ΤΗΝ ΚΑΖΕΙΝΗ, ΚΑΙ (V) ΤΑ ΣΥΝΘΕΤΙΚΑ ΦΩΣΦΟΠΕΠΤΙΔΙΑ RRATVA ΚΑΙ RRREEETEEEAA ΤΑ ΟΠΟΙΑ ΕΙΝΑΙ ΔΙΑΠΙΣΤΩΜΕΝΑ ΥΠΟΣΤΡΩΜΑΤΑ ΓΙΑ ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ, ΟΧΙ ΟΜΩΣ ΚΑΙ ΓΙΑ ΜΗ ΕΙΔΙΚΕΣ ΟΞΙΝΕΣ ΚΑΙ ΑΛΚΑΛΙΚΕΣ ΦΩΣΦΑΤΑΣΕΣ. Η ΜΙΚΡΟΚΥΣΤΙΝΗ LR, ΕΝΑΣ ΙΣΧΥΡΟΣ ΑΝΑΣΤΟΛΕΑΣ ΤΩΝ PP-1 ΚΑΙ PP-2A, ΑΝΑΣΤΕΛΛΕΙ ΔΡΑΣΤΙΚΑ ΤΙΣ PVP1ΚΑΙ PVP2. ΟΙ PVP1 ΚΑΙ PVP2 ΑΠΟ ΕΓΚΕΦΑΛΟ ΑΡΟΥΡΑΙΟΥ ΠΑΡΟΥΣΙΑΖΟΥΝ ΜΟΡΙΑΚΑ ΚΑΙ ΒΙΟΧΗΜΙΚΑ ΧΑΡΑΚΤΗΡΙΣΤΙΚΑ ΠΟΥ ΔΙΑΦΕΡΟΥΝ ΑΠΟ ΤΙΣ ΓΝΩΣΤΕΣ ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ.ΜΕΛΕΤΗΣΑΜΕ ΤΗΝ ΕΠΙΔΡΑΣΗ ΤΩΝ ΙΟΝΤΩΝ ΤΟΥ ΑΡΓΙΛΙΟΥ ΣΤΗΝ ΑΠΟΦΩΣΦΟΡΥΛΙΩΣΗ ΤΗΣ ΦΩΣΒΙΤΙΝΗΣ ΠΟΥ ΚΑΤΑΛΥΕΤΑΙ ΑΠΟ ΟΞΙΝΗ ΦΩΣΦΑΤΑΣΗ, ΣΤΗ ΜΕΤΑΧΡΩΜΑΤΙΚΗ ΑΝΤΙΔΡΑΣΗ, ΚΑΙ ΣΤΗΝ ΗΛΕΚΤΡΟΦΟΡΗΤΙΚΗ ΚΙΝΗΤΙΚΟΤΗΤΑ ΤΗΣ ΦΩΣΒΙΤΙΝΗΣ. (ΠΕΡΙΚΟΠΗ ΠΕΡΙΛΗΨΗΣ)

THE MECHANISM OF DEPHOSPHORYLATION OF MUTIPHOSPHORYLATED PROTEINS IN THE MAMMALIAN BRAIN IS NOT WELL UNDERSTOOD. WE HAVE USED THE ENDOGENOUSLY MULTIPHOSPHORYLATED PROTEIN, PHOSVITIN, AS A MODEL SUBSTRATE AND UNDERTAKEN THE PURIFICATIONAND CHARACTERIZATION OF BRAIN PHOSPHATASES THAT PREFERENTIALLY DEPHOSPHORYLATE MULTIPHOSPHORYLATED PROTEINS. WE HAVE DEVELOPED A SIMPLE AND SENSITIVE COLORIMETRIC ASSAY FOR PROTEIN PHOSPHATASE ACTIVITY USING A MALACHITE GREEN PROCEDURE. TWO PHOSVITIN PHOSPHATASE ACTIVITIES, TERMED PHOSVITIN PHOSPHATASE 1 AND2 (PVP1 & PVP2), WERE ISOLATED FROM RAT BRAIN AND CHARACTERIZED. BOTH PHOSPHATASES DEPHOSPHORYLATE ALSO (I) PHOSPHORYLASE A, (II) THE Α AND Β SUBUNITS OF PHOSPHORYLASE KINASE, (III) THE MICROTUBULE-ASSOCIATED PROTEIN TAU (Τ), (IV) CASEIN, AND (V) THE SYNTHETIC PHOSPHOPEPTIDES RRATVA AND RRREEETEEEAA WHICH ARE DEFINED SUBSTRATES FOR PROTEIN PHOSPHATASES BUT NOT FOR NONSPECIFIC ACID OR ALKALINE PHOSPHATASES. MICROCYSTIN LR, A POTENT ENZYME-DIRECTED INHIBITOR OF PP-1AND PP-2A, DRASTICALLY INHIBITS PVP1 AND PVP2. BRAIN PVP1 AND PVP2 DISPLAY MOLECULAR AND BIOCHEMICAL CHARACTERISTICS DISTINCT FROM THE KNOWN PROTEIN PHOSPHATASES. THE EFFECTS OF ALUMINUM IONS ON THE DEPHOSPHORYLATION OF PHOSVITIN CATALYZED BY ACID PHOSPHATASE, ON METACHROMASIA, AND ON THE ELECTROPHORETIC MOBILITY OF PHOSVITIN WERE ALSO STUDIED. IN ALL CASES THE ACTION OF AL3+ WAS INHIBITORY IN A CONCENTRATION- AND TIME-DEPENDENT MANNER.

Subjects/Keywords: ALUMINUM ACTION; Brain; MALACHITE GREEN; MULTIPHOSPHORYLATED PROTEINS; PHOSPHATE DETERMINATION; PHOSVITIN; PROTEIN PHOSPHATASES; TAU DEPHOSPHORYLATION; ΑΠΟΦΩΣΦΟΡΥΛΙΩΣΗ ΤΗΣ ΤΑΥ; ΔΡΑΣΗ ΑΡΓΙΛΙΟΥ; Εγκέφαλος; ΠΟΛΥΦΩΣΦΟΡΥΛΙΩΜΕΝΕΣ ΠΡΩΤΕΙΝΕΣ; ΠΡΑΣΙΝΟ ΤΟΥ ΜΑΛΑΧΙΤΗ; ΠΡΟΣΔΙΟΡΙΣΜΟΣ ΦΩΣΦΟΡΙΚΩΝ; ΦΩΣΒΙΤΙΝΗ; ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

ΓΕΛΑΔΟΠΟΥΛΟΣ, . (1996). ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΣΕ ΕΓΚΕΦΑΛΟ ΘΗΛΑΣΤΙΚΩΝ. (Thesis). Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών (ΕΚΠΑ); National and Kapodistrian University of Athens. Retrieved from http://hdl.handle.net/10442/hedi/8135

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

ΓΕΛΑΔΟΠΟΥΛΟΣ, ΤΑΞΙΑΡΧΗΣ. “ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΣΕ ΕΓΚΕΦΑΛΟ ΘΗΛΑΣΤΙΚΩΝ.” 1996. Thesis, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών (ΕΚΠΑ); National and Kapodistrian University of Athens. Accessed July 19, 2019. http://hdl.handle.net/10442/hedi/8135.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

ΓΕΛΑΔΟΠΟΥΛΟΣ, ΤΑΞΙΑΡΧΗΣ. “ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΣΕ ΕΓΚΕΦΑΛΟ ΘΗΛΑΣΤΙΚΩΝ.” 1996. Web. 19 Jul 2019.

Vancouver:

ΓΕΛΑΔΟΠΟΥΛΟΣ . ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΣΕ ΕΓΚΕΦΑΛΟ ΘΗΛΑΣΤΙΚΩΝ. [Internet] [Thesis]. Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών (ΕΚΠΑ); National and Kapodistrian University of Athens; 1996. [cited 2019 Jul 19]. Available from: http://hdl.handle.net/10442/hedi/8135.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

ΓΕΛΑΔΟΠΟΥΛΟΣ . ΦΩΣΦΑΤΑΣΕΣ ΤΩΝ ΠΡΩΤΕΙΝΩΝ ΣΕ ΕΓΚΕΦΑΛΟ ΘΗΛΑΣΤΙΚΩΝ. [Thesis]. Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών (ΕΚΠΑ); National and Kapodistrian University of Athens; 1996. Available from: http://hdl.handle.net/10442/hedi/8135

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

.