5c). quick 3D match on all the 50,000 proteins in the PDB database. It is to be noted the 4 collapse enrichment achieved on this set is an underestimation of the power of our method since the chosen set of proteins was a filtered category through 3D congruence.(PDF) pone.0028470.s002.pdf (74K) GUID:?9437DE58-7399-44F2-A339-BABD3BEFBACA Number S3: The overall pathway for -lactam hydrolysis. You will find two main methods – acylation and hydrolysis. The acylation is definitely common to -lactamases and penicillin-binding proteins (PBP). Therefore antibiotic resistance essentially arises from the deacylation reaction. Starting with the ground state (a) and moving through a high energy acylation state (b) the acyl-enyzme intermediate is definitely formed (c) due to the nucleophilic assault of the serine within the -lactam. The next step is hydrolysis (d) and finally the product is definitely formed (e), and the enzyme is definitely ready for another cycle.(PDF) pone.0028470.s003.pdf (110K) GUID:?D1B2BF23-6DC1-4EAF-9FA9-D659CFF9F731 Number S4: The potential difference between the Ser and Lys residues in all the SXXK motifs (in a sample of 1500 motifs where each one is numbered arbitrarily) found in the 3000 non-redundant proteins.(PDF) pone.0028470.s004.pdf (123K) GUID:?85767394-EA93-443C-BD66-C2F544289C18 Figure S5: MALDI mass spectra of the purified SAP. The small maximum at approximately 28 kDa represents the doubly charged molecular ion at half the m/z value.(PDF) pone.0028470.s005.pdf (42K) GUID:?F62C61EB-F632-40A4-831D-53C096CBF819 Figure S6: Protease activity of SAP after purification. Substrate protein (UVI31+; lane 1) was incubated over night at 37C with stock SAP (lane 2) and purified SAP (lane 3). Purification was carried out by moving the protein through a 50 kDA centrifugal filter device followed by gel elution of a single polypeptide band related to the size of SAP from a 8% native PAGE gel by electroelution (Centrilutor micro-electroelutor from Millipore).(PDF) pone.0028470.s006.pdf (66K) GUID:?FDFA7C9C-CE03-40D3-94A2-8D0210A2AE85 Figure S7: Algorithm ScoreSingleProtein – score any given protein for enzymatic function (PDB id: 1P1M) (CLASP predicted residues: His55,Glu203,Asp279,Asp113).(PDF) pone.0028470.s012.pdf (52K) GUID:?3746B13A-C564-4CA8-9139-F67502DE8CDC Table S5: Identity/Similarity among all Aps.(PDF) pone.0028470.s013.pdf (57K) GUID:?1370ACBA-9B65-42EC-B5C1-69960F139963 Table S6: Potential difference between Lys73 and Glu166 for any motif from a Class A -lactamase which now includes the Glu166 for a list of Class A -lactamase proteins Ser70, Lys73, Ser130, Lys234, Glu166, the high potential differences observed are consistent with the theory that Lys73 is definitely protonated in the initial stages, and acts as the general base to Ser70 only after transferring a proton to the Glu166.(PDF) pone.0028470.s014.pdf (29K) GUID:?218F0D5A-89BE-4CA3-817F-F9DB8FF7D368 Table S7: Dataset.(PDF) pone.0028470.s015.pdf (69K) GUID:?A8D3B828-78DA-4E90-8EE5-1A777ACEC71C Abstract Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural info that supplement sequence conservation centered analyses. Development of tools that compute electrostatic potentials offers further improved our ability to better characterize the active site residues in proteins. We have explained a computational strategy for detecting active sites based on structural and electrostatic conformity – C deviations from standard values, we display that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are related. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are Rabbit Polyclonal to MRGX3 declined. We 1st present the results of active site prediction by CLASP for two enzymatic activities – -lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) will also be presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for more activities. Such a search offers led us to forecast a hitherto unfamiliar function of shrimp alkaline phosphatase (SAP), where the protein functions as a protease. Finally, we present.We 1st present the results of active site prediction by CLASP for two enzymatic activities – -lactamases and serine proteases, two of the most extensively investigated enzymes. chosen set of proteins was a filtered category through 3D congruence.(PDF) pone.0028470.s002.pdf (74K) GUID:?9437DE58-7399-44F2-A339-BABD3BEFBACA Number S3: The overall pathway for -lactam hydrolysis. You will find two main methods – acylation and hydrolysis. The acylation is definitely common to -lactamases and penicillin-binding proteins (PBP). Therefore antibiotic resistance essentially arises from the deacylation reaction. Starting with the ground state (a) and moving through a high energy acylation state (b) the acyl-enyzme intermediate is definitely formed (c) due to the nucleophilic assault of the serine within the -lactam. The next step is hydrolysis (d) and finally the product is definitely formed (e), and the enzyme is definitely ready for another cycle.(PDF) pone.0028470.s003.pdf (110K) GUID:?D1B2BF23-6DC1-4EAF-9FA9-D659CFF9F731 Number S4: The Aclidinium Bromide potential difference between the Ser and Lys residues in all the SXXK motifs (in a sample of 1500 motifs where each one is numbered arbitrarily) found in the 3000 non-redundant proteins.(PDF) pone.0028470.s004.pdf Aclidinium Bromide (123K) GUID:?85767394-EA93-443C-BD66-C2F544289C18 Figure S5: MALDI mass spectra of the purified SAP. The small peak at approximately 28 kDa represents the doubly charged molecular ion at half the m/z value.(PDF) pone.0028470.s005.pdf (42K) GUID:?F62C61EB-F632-40A4-831D-53C096CBF819 Figure S6: Protease activity of SAP after purification. Substrate protein (UVI31+; lane 1) was incubated over night at 37C with stock SAP (lane 2) and purified SAP (lane 3). Purification was carried out by moving the protein through a 50 kDA centrifugal filter device followed by gel elution of a single polypeptide band related to the size of SAP from a 8% native PAGE gel by electroelution (Centrilutor micro-electroelutor from Millipore).(PDF) pone.0028470.s006.pdf (66K) GUID:?FDFA7C9C-CE03-40D3-94A2-8D0210A2AE85 Figure S7: Algorithm ScoreSingleProtein – score any given protein for enzymatic function (PDB id: 1P1M) (CLASP predicted residues: His55,Glu203,Asp279,Asp113).(PDF) pone.0028470.s012.pdf (52K) GUID:?3746B13A-C564-4CA8-9139-F67502DE8CDC Table S5: Identity/Similarity among all Aps.(PDF) pone.0028470.s013.pdf (57K) GUID:?1370ACBA-9B65-42EC-B5C1-69960F139963 Table S6: Potential difference between Lys73 and Glu166 for any motif from a Class A -lactamase which now includes the Glu166 for a list of Class A -lactamase proteins Ser70, Lys73, Ser130, Lys234, Glu166, the high potential differences observed are consistent with the theory that Lys73 is definitely protonated in the initial stages, and acts as the general base to Ser70 only after transferring a proton to the Glu166.(PDF) pone.0028470.s014.pdf (29K) GUID:?218F0D5A-89BE-4CA3-817F-F9DB8FF7D368 Table S7: Dataset.(PDF) pone.0028470.s015.pdf (69K) GUID:?A8D3B828-78DA-4E90-8EE5-1A777ACEC71C Abstract Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural info that supplement sequence conservation centered analyses. Development of tools that compute electrostatic potentials offers further improved our ability to better characterize the active site residues in proteins. Aclidinium Bromide We have explained a computational strategy for detecting active sites based on structural and electrostatic conformity – C deviations from standard values, we display that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are related. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are declined. We 1st present the results of active site prediction by CLASP for two enzymatic activities – -lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) will also be presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for more activities. Such a search offers led us to forecast a hitherto unfamiliar function of shrimp alkaline phosphatase (SAP), where the protein functions as a protease. Finally, we.