[ccp4bb] AMP-PNP Hydrolysis
Hi there, Was recently looking at a structure of an enzyme with AMP-PNP added to the crystallization mix, and all I see is density for ADP. I was wondering if hydrolysis of AMP-PNP to ADP is relatively common - either as a result of extended time in crystallization or exposure of the resultant crystals to synchrotron radiation? I know that there can be up to 10% contamination of ADP in the purchased material, so it could just be that we have selected that form in the crystal, or that there was endogenous ADP bound that failed to substitute. Just curious if hydrolysis is a common observation. Thanks in advance- Steve Stephen M. Soisson, Ph.D. Structural Chemistry Site Lead, WP Merck Research Laboratories 770 Sumneytown Pike, WP14-1101 West Point, PA 19486 Phone: (215) 652-6185 Fax:(215) 652-9051 stephen_sois...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] AMP-PNP Hydrolysis
Hi Steve, With my experience, it is (very) common to see AMPPNP is hydrolyzed to AMPPN (supposedly) with my protein. Although the literature often reported AMPPNP as a stable ATP mimic, such a luck wasn't true with my case, maybe same as you. If you go to Sigma website where I purchased, it may say it is not stable in an acidic condition. My mother liquor was in an acidic condition. So you'd better consider if you used it in an acidic condition, otherwise, your protein inherently has a strong power to hydrolyze it. In addition to the pH, I often see it can go hydrolysis easily. However, you can try more as you mentioned it may contain impurity. I just want to inform you that it is not surprising to see this hydrolysis. Good luck~ Young-Jin On Mon, Feb 14, 2011 at 8:30 AM, Soisson, Stephen M stephen_sois...@merck.com wrote: Hi there, Was recently looking at a structure of an enzyme with AMP-PNP added to the crystallization mix, and all I see is density for ADP. I was wondering if hydrolysis of AMP-PNP to ADP is relatively common - either as a result of extended time in crystallization or exposure of the resultant crystals to synchrotron radiation? I know that there can be up to 10% contamination of ADP in the purchased material, so it could just be that we have selected that form in the crystal, or that there was endogenous ADP bound that failed to substitute. Just curious if hydrolysis is a common observation. Thanks in advance- Steve Stephen M. Soisson, Ph.D. *Structural Chemistry Site Lead, WP* Merck Research Laboratories 770 Sumneytown Pike, WP14-1101 West Point, PA 19486 Phone: (215) 652-6185 Fax:(215) 652-9051 stephen_sois...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] AMP-PNP Hydrolysis
Hi Steve, Funnily enough I just read the following paper today, which describes exactly this phenomenon: http://www.ncbi.nlm.nih.gov/pubmed/21093442 Is AMPPCP as sensitive to acid conditions? I would suspect not. Best wishes Derek ___ Derek Logantel: +46 46 222 1443 Associate Professorfax: +46 46 222 4692 Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 Centre for Molecular Protein Science www.cmps.lu.sehttp://www.cmps.lu.se Lund University, Box 124, 221 00 Lund, Sweden www.saromics.comhttp://www.saromics.com On Feb 14, 2011, at 15:05, Young-Jin Cho wrote: Hi Steve, With my experience, it is (very) common to see AMPPNP is hydrolyzed to AMPPN (supposedly) with my protein. Although the literature often reported AMPPNP as a stable ATP mimic, such a luck wasn't true with my case, maybe same as you. If you go to Sigma website where I purchased, it may say it is not stable in an acidic condition. My mother liquor was in an acidic condition. So you'd better consider if you used it in an acidic condition, otherwise, your protein inherently has a strong power to hydrolyze it. In addition to the pH, I often see it can go hydrolysis easily. However, you can try more as you mentioned it may contain impurity. I just want to inform you that it is not surprising to see this hydrolysis. Good luck~ Young-Jin On Mon, Feb 14, 2011 at 8:30 AM, Soisson, Stephen M stephen_sois...@merck.commailto:stephen_sois...@merck.com wrote: Hi there, Was recently looking at a structure of an enzyme with AMP-PNP added to the crystallization mix, and all I see is density for ADP. I was wondering if hydrolysis of AMP-PNP to ADP is relatively common - either as a result of extended time in crystallization or exposure of the resultant crystals to synchrotron radiation? I know that there can be up to 10% contamination of ADP in the purchased material, so it could just be that we have selected that form in the crystal, or that there was endogenous ADP bound that failed to substitute. Just curious if hydrolysis is a common observation. Thanks in advance- Steve Stephen M. Soisson, Ph.D. Structural Chemistry Site Lead, WP Merck Research Laboratories 770 Sumneytown Pike, WP14-1101 West Point, PA 19486 Phone: (215) 652-6185 Fax:(215) 652-9051 stephen_sois...@merck.commailto:stephen_sois...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] AMP-PNP Hydrolysis
Hello Steve, You can also check out this paper: Bystrom, Pettigrew, Remington and Branchaud (1997) Bioorganic Medicinal Chemistry Letters, Vol 7 No 20 pp2613-2616. It describes the creation of AMPPCF2P, which I had opportunity to use a few years back and it worked great! Good luck, Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Derek Logan Sent: Monday, February 14, 2011 4:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AMP-PNP Hydrolysis Hi Steve, Funnily enough I just read the following paper today, which describes exactly this phenomenon: http://www.ncbi.nlm.nih.gov/pubmed/21093442 Is AMPPCP as sensitive to acid conditions? I would suspect not. Best wishes Derek ___ Derek Logantel: +46 46 222 1443 Associate Professorfax: +46 46 222 4692 Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 Centre for Molecular Protein Science www.cmps.lu.se Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com On Feb 14, 2011, at 15:05, Young-Jin Cho wrote: Hi Steve, With my experience, it is (very) common to see AMPPNP is hydrolyzed to AMPPN (supposedly) with my protein. Although the literature often reported AMPPNP as a stable ATP mimic, such a luck wasn't true with my case, maybe same as you. If you go to Sigma website where I purchased, it may say it is not stable in an acidic condition. My mother liquor was in an acidic condition. So you'd better consider if you used it in an acidic condition, otherwise, your protein inherently has a strong power to hydrolyze it. In addition to the pH, I often see it can go hydrolysis easily. However, you can try more as you mentioned it may contain impurity. I just want to inform you that it is not surprising to see this hydrolysis. Good luck~ Young-Jin On Mon, Feb 14, 2011 at 8:30 AM, Soisson, Stephen M stephen_sois...@merck.com wrote: Hi there, Was recently looking at a structure of an enzyme with AMP-PNP added to the crystallization mix, and all I see is density for ADP. I was wondering if hydrolysis of AMP-PNP to ADP is relatively common - either as a result of extended time in crystallization or exposure of the resultant crystals to synchrotron radiation? I know that there can be up to 10% contamination of ADP in the purchased material, so it could just be that we have selected that form in the crystal, or that there was endogenous ADP bound that failed to substitute. Just curious if hydrolysis is a common observation. Thanks in advance- Steve Stephen M. Soisson, Ph.D. Structural Chemistry Site Lead, WP Merck Research Laboratories 770 Sumneytown Pike, WP14-1101 West Point, PA 19486 Phone: (215) 652-6185 Fax:(215) 652-9051 stephen_sois...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] AMP-PNP Hydrolysis
My five eurocents to the discussion ... AMPPNP can also be specifically hydrolyzed by Serca1a, the Sarcoplasmic Reticulum Ca2+-ATPase. Such cleavage was proven to be calcium-dependent meaning that AMPPNP is used by the ATPase as a genuine substrate although with a very slow kinetics compared to what is obtained with the cognate substrate ATP. J Biol Chem. 1981 Oct 10;256(19):9793-5. Sarcoplasmic reticulum ATPase catalyzes hydrolysis of adenyl-5'-yl imidodiphosphate. Taylor JS. Abstract Sarcoplasmic reticulum ATPase has been found to cleave the ATP analog adenyl-5'-yl imidodiphosphate in a calcium-dependent reaction. The reaction products were determined by 31P NMR to be inorganic phosphate and adenyl-5'-yl phosphoramidate (AMP-PN). AMP-PNP hydrolysis, like ATP hydrolysis, drives active Ca2+ accumulation by sarcoplasmic reticulum vesicles. PMID: 6456267 [PubMed - indexed for MEDLINE] This property allowed us to indeed trap and cystallize the protein under an AMPPN-bound, phosphoenzyme intermediate, that was proposed to be a fair mimick of the so-called E1P (Ca2+).ADP state. Display Settings:AbstractSend to: Nature. 2007 Dec 13;450(7172):1036-42. The structural basis of calcium transport by the calcium pump. Olesen C, Picard M, Winther AM, Gyrup C, Morth JP, Oxvig C, Møller JV, Nissen P. Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, University of Aarhus, Ole Worms Alle, blg. 1185, DK - 8000 Aarhus C, Denmark. Comment in: Nature. 2007 Dec 13;450(7172):957-9. Abstract The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical gradients required for key functions in eukaryotic cells. PMID: 18075584 [PubMed - indexed for MEDLINE] Martin Picard Le 14 févr. 2011 à 22:53, Prince, D Bryan dbryan.pri...@astrazeneca.com a écrit : Hello Steve, You can also check out this paper: Bystrom, Pettigrew, Remington and Branchaud (1997) Bioorganic Medicinal Chemistry Letters, Vol 7 No 20 pp2613-2616. It describes the creation of AMPPCF2P, which I had opportunity to use a few years back and it worked great! Good luck, Bryan Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Derek Logan Sent: Monday, February 14, 2011 4:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AMP-PNP Hydrolysis Hi Steve, Funnily enough I just read the following paper today, which describes exactly this phenomenon: http://www.ncbi.nlm.nih.gov/pubmed/21093442 Is AMPPCP as sensitive to acid conditions? I would suspect not. Best wishes Derek ___ Derek Logantel: +46 46 222 1443 Associate Professorfax: +46 46 222 4692 Dept. of Biochemistry and Structural Biology mob: +46 76 8585 707 Centre for Molecular Protein Science www.cmps.lu.se Lund University, Box 124, 221 00 Lund, Sweden www.saromics.com On Feb 14, 2011, at 15:05, Young-Jin Cho wrote: Hi Steve, With my experience, it is (very) common to see AMPPNP is hydrolyzed to AMPPN (supposedly) with my protein. Although the literature often reported AMPPNP as a stable ATP mimic, such a luck wasn't true with my case, maybe same as you. If you go to Sigma website where I purchased, it may say it is not stable in an acidic condition. My mother liquor was in an acidic condition. So you'd better consider if you used it in an acidic condition, otherwise, your protein inherently has a strong power to hydrolyze it. In addition
Re: [ccp4bb] AMP-PNP Hydrolysis
Is it possible that the phosphates are just disordered rather than being cleaved? It's always the case for inactive kinase-ATP or AMPPNP complexes that the phosphates are not stabilized by Mg2+ or the residues in the binding pocket and hence they become disordered and are not seen in the electron density. It may be worth to take a look at those phosphate-binding residues in your enzyme and see if they are positioned towards your AMPPNP's phosphates. HTH, Matt On Mon, Feb 14, 2011 at 5:30 AM, Soisson, Stephen M stephen_sois...@merck.com wrote: Hi there, Was recently looking at a structure of an enzyme with AMP-PNP added to the crystallization mix, and all I see is density for ADP. I was wondering if hydrolysis of AMP-PNP to ADP is relatively common - either as a result of extended time in crystallization or exposure of the resultant crystals to synchrotron radiation? I know that there can be up to 10% contamination of ADP in the purchased material, so it could just be that we have selected that form in the crystal, or that there was endogenous ADP bound that failed to substitute. Just curious if hydrolysis is a common observation. Thanks in advance- Steve Stephen M. Soisson, Ph.D. *Structural Chemistry Site Lead, WP* Merck Research Laboratories 770 Sumneytown Pike, WP14-1101 West Point, PA 19486 Phone: (215) 652-6185 Fax:(215) 652-9051 stephen_sois...@merck.com Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432
[ccp4bb] Fwd: [ccp4bb] AMP-PNP Hydrolysis
Once again, I've sent off a message only to one person that was intended for the BB. Begin forwarded message: From: Charles W. Carter, Jr car...@med.unc.edu Date: February 14, 2011 6:23:16 PM EST To: Martin Picard martin.picard...@gmail.com Subject: Re: [ccp4bb] AMP-PNP Hydrolysis I take a similar viewpoint to Martin's. The hydrolysis of ATP analogs was reviewed carefully by Ralph Yount who did the early work on AMP-PNP (Biochemistry 1971, 10:2484) and he reviewed the field in a 1975 paper in Advances in Enzymology (43:1-57). A remarkably widely ignored paper on the use of AMP-PNP is: Marston, S. B., Tregear, R. T., Bodger, C. D. Clarke, M. L. (1979). Coupling Between the Enzymatic Site of Myosin and the Mechanical Output of Muscle. J. Mol. Biol. 128, 111-126. In this paper, the authors show that glycerinated muscle fibers in Rigor undergo a rapid, reversible, and stress-independent increase in rest length equivalent to about half to three quarters of a cross bridge motion when treated with AMPPNP. This happens without loss of stiffness. The only conclusion I can draw from the abstract of this paper, which I've summarized here, is that the analog is very rapidly hydrolyzed, presumeably by myosin, and without dissociation of the rigor complexes. The hydrolysis products are far less soluble in water than their homologous products from ATP hydrolysis, according to splendid experiments done years ago by Dick Wolfenden. This puts myosin in the unusual position of having to adapt to bound products. Its only recourse is to adopt the 90 degree attitude toward actin, thus increasing the rest length by the just the right amount. In this experiment, AMP-PNP is acting like a hydrolysis products analog. The rate of hydrolysis, in situ, must be very rapid; otherwise the rigor complexes would dissociate. I've discussed this with Mike Reedy at Duke, and I think he agreed with this interpretation. It is a stupendous demonstration of macroscopic reversibility of the myosin cross-bridge cycle. My interpretation, together with Martin's reference to the Ca pump protein suggest that perhaps a massive amount of structural biology and biochemical experimentation on adenine and guanine imido-phosphates has been misinterpreted. Charlie On Feb 14, 2011, at 5:34 PM, Martin Picard wrote: My five eurocents to the discussion ... AMPPNP can also be specifically hydrolyzed by Serca1a, the Sarcoplasmic Reticulum Ca2+-ATPase. Such cleavage was proven to be calcium-dependent meaning that AMPPNP is used by the ATPase as a genuine substrate although with a very slow kinetics compared to what is obtained with the cognate substrate ATP. J Biol Chem. 1981 Oct 10;256(19):9793-5. Sarcoplasmic reticulum ATPase catalyzes hydrolysis of adenyl-5'-yl imidodiphosphate. Taylor JS. Abstract Sarcoplasmic reticulum ATPase has been found to cleave the ATP analog adenyl-5'-yl imidodiphosphate in a calcium-dependent reaction. The reaction products were determined by 31P NMR to be inorganic phosphate and adenyl-5'-yl phosphoramidate (AMP-PN). AMP-PNP hydrolysis, like ATP hydrolysis, drives active Ca2+ accumulation by sarcoplasmic reticulum vesicles. PMID: 6456267 [PubMed - indexed for MEDLINE] This property allowed us to indeed trap and cystallize the protein under an AMPPN-bound, phosphoenzyme intermediate, that was proposed to be a fair mimick of the so-called E1P (Ca2+).ADP state. Display Settings:AbstractSend to: Nature. 2007 Dec 13;450(7172):1036-42. The structural basis of calcium transport by the calcium pump. Olesen C, Picard M, Winther AM, Gyrup C, Morth JP, Oxvig C, Møller JV, Nissen P. Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, University of Aarhus, Ole Worms Alle, blg. 1185, DK - 8000 Aarhus C, Denmark. Comment in: Nature. 2007 Dec 13;450(7172):957-9. Abstract The sarcoplasmic reticulum Ca2+-ATPase, a P-type ATPase, has a critical role in muscle function and metabolism. Here we present functional studies and three new crystal structures of the rabbit skeletal muscle Ca2+-ATPase, representing the phosphoenzyme intermediates associated with Ca2+ binding, Ca2+ translocation and dephosphorylation, that are based on complexes with a functional ATP analogue, beryllium fluoride and aluminium fluoride, respectively. The structures complete the cycle of nucleotide binding and cation transport of Ca2+-ATPase. Phosphorylation of the enzyme triggers the onset of a conformational change that leads to the opening of a luminal exit pathway defined by the transmembrane segments M1 through M6, which represent the canonical membrane domain of P-type pumps. Ca2+ release is promoted by translocation of the M4 helix, exposing Glu 309, Glu 771 and Asn 796 to the lumen. The mechanism explains how P-type ATPases are able to form the steep electrochemical
