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 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 Logan tel: +46 46 222 1443 >>> Associate Professor fax: +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. >>> >>> >>> >>> >>> >>> >>> >>> >>> >>> >>> >>> >
