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='''<font color = 'black'> A Physical Model of the β2-Adrenergic Receptor</font>'''=
<applet load='aln_1H6M_to_1HEW_2.pdb' size='300' frame='true' align='right' caption=' Hen Egg White (HEW) Lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to the active site, PDBid 1HEW' scene='User:Judy_Voet/Lysozyme/Lysozyme1/16' />
 
Lysozyme was the first enzyme whose X-ray structure was determined <ref> PMID 5840126</ref><ref>Phillips, D. C. The hen egg white lysozyme molecule. Proc. Natl Acad. Sci. USA 57, 483-495 (1967)</ref>. This <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>scene </scene>  shows Hen Egg White (HEW) lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to a cleft in the enzyme. David Phillips, who determined the structure in 1965, saw that the cleft was large enough to fit three more saccharide units. He therefore built a model extending the trisaccharide to a 
=='''<font color = 'red'>A SMART Team Molecular Story</font><font color = 'black'> --- from the Madison West High School 2008 SMART Team</font>'''==
<scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> that fits into the cleft, labeling the sugar subsites A-F<ref> coordinates of the model kindly provided by Louise Johnson</ref>. Alternately click on <scene name='User:Judy_Voet/Lysozyme/Lysozyme1/15'>trisaccharide</scene> and <scene name='User:Judy_Voet/Lysozyme/Lysozyme1_hexamer/7'>hexasaccharide</scene> to turn the modeled portion of the hexasaccharide on and off.
 
The interesting thing about the model was that the only way that the hexasaccharide would fit into the cleft was if the 4th saccharide (in subsite D) was strained into a <scene name='User:Judy_Voet/Lysozyme/Half-chair/2'>half-chair conformation</scene>. This conformation is what would be necessary for the formation of an oxocarbenium ion (oxionium ion). When the model was studied, <scene name='User:Judy_Voet/Lysozyme/Glu_35/1'>Glu 35</scene> was found to be in an ideal location to act as a general acid catalyst, 3.34 Angstroms from the bridging oxygen between the 4th and 5th saccharide units. <scene name='User:Judy_Voet/Lysozyme/Asp_52/2'>Asp 52</scene> appeared to be too far away (2.69 angstroms) in the static lysozyme structure to have formed a covalent bond with C1 of the half-chair model in the D site, and no covalent intermediate had ever been detected, so Phillips proposed that it acted as an electrostatic stabilizer of the oxonium ion (referred to as The Phillips Mechanism).
:Students -- Dianna Amasino, Axel Glaubitz, Susan Huang, Joy Li, Hsien-Yu Shih, Junyao Song, Esther Yoon, Xiao Zhu:
 
:Advisor: Basudeb Bhattacharyya / Assistant: Peter Vander Velden
 
:Mentors: David Nelson, Ph.D. and Jim Keck, Ph.D., University of Wisconsin-Madison, Madison, WI
 
 
 
 
<applet load='2rh1.pdb' size='350' frame='true'  align='left' scene='Hoelzer_Sandbox/First_image/1'/>
<swf width="550" height="380">http://myweb.msoe.edu/~hoelzer/proteopdedia.swf</swf>
 
 
{{clear}}
 
==<font color = 'blue'>Abstract for Our Project</font>==
 
G protein-coupled receptors (GPCRs) are the largest family of integral membrane proteins coded by the human genome. GPCRs are important for signal transduction and share the general structural characteristic of a plasma membrane receptor with seven transmembrane segments.  More than 50% of human therapeutics act on a member of the GPCR family of proteins. One example of a GPCR targeted by pharmaceutical companies is the β2-adrenergic receptor.  Adrenergic receptors are found throughout the body and are triggered by the hormone epinephrine (also known as adrenaline, hence the name adrenergic).  When epinephrine binds to the receptor, it causes a slight conformational change within the receptor.  This change then triggers activation of a G-protein --- proteins that bind GTP and are coupled to the receptor on the cytoplasmic side of the receptor --- causing dissociation of the G-protein from the receptor. Through the transfer of GTP, G-protein activates an enzyme that converts ATP into cyclic AMP, which induces a response within the cell (for example, muscle contraction if the receptor is located on a muscle cell). When this signal transduction event functions normally in the body, it helps regulate heart rate and blood pressure and is important for the “fight or flight” response.  It is important medically to be able to manipulate these functions in cases of high blood pressure or heart failure through the use of beta blockers, a medicine designed to bind to adrenergic receptors, thus inhibiting the binding of epinephrine, and resulting in a lack of effect of the hormone on the body.  We have used rapid prototyping technology to model the interaction of the human β2-adrenergic receptor with the beta blocker, carazolol.  The structure is dominated by seven alpha helices and is representative of the structure of GPCRs.  By modeling the β2-adrenergic receptor, we hope to better understand GPCRs as well as understand the mechanism of hormone/drug binding, which will aid in developing better drug treatments.


{{Clear}}
{{Clear}}


==<font color = 'blue'>Creating the Physical Model of the β2-adrenergic receptor</font>==
<applet load='aln_1H6M_to_1HEW_2.pdb' size='300' frame='true' align='right' caption='NAG-2-deoxy-2-fluoro-glucosyl fluoride (NAG2FGlcF) bound to Glu35Gln HEW Lysozyme PDBid 1H6M' scene='User:Judy_Voet/Lysozyme/1h6m/3'/>
 
Then, in 2001, Stephen Withers published <scene name='User:Judy_Voet/Lysozyme/1h6m/3'>1H6M</scene>,<ref>PMID 11518970</ref> in which Glu 35 he had mutated to Gln to remove the general acid catalyst. The substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF). The fluoro group on C-1 does not require acid catalysis to be a good leaving group, and the remaining saccharide, in the absence of the acid necessary to catalyse the second step of the reaction, was demonstrated to form a <scene name='User:Judy_Voet/Lysozyme/Covalent/1'> covalent intermediate</scene>. In this <scene name='User:Judy_Voet/Lysozyme/Superposition/2'>superposition</scene> of the half chair model with 1HEW (greens) and the covalent intermediate in 1H6M (blues), note  the relatively small motions of Asp 52 and C1 of the sugar ring in going from the model to the covalent intermediate. to observe the motion from the <scene name='User:Judy_Voet/Lysozyme/Asp52_halfchair/1'>half-chair</scene> to the <scene name='User:Judy_Voet/Lysozyme/Covalent/2'>covalent intermediate</scene> just toggle between the two green links.  
<applet load='2rh1.pdb' size='400' frame='true' align='left' caption='Madison West SMART Team Model - β2-Adrenergic Receptor' scene='Hoelzer_Sandbox/Building_our_model/8'/>
===Some Useful External Links===
 
[http://en.wikipedia.org/wiki/Lysozyme Lysozyme]
 
Virtually any image of a protein that can be created in the computer environment of RP-RasMol, can be converted into a physical model of the protein using rapid prototyping technology.  To design our model of the β2-adrenergic receptor, we used the atomic coordinates for this structure as reported in the pdb file 2rh1, from the Ray Stevens laboratory at the Scripps Research Institute. 
 
--- Our model represents <scene name='Hoelzer_Sandbox/Image_1/1'>amino acids 29-230 and 263-342 </scene>. 
 
--- Starting with a cpk-colored, spacefilled representation of the protein, we simplified this image by converting it to an <scene name='Hoelzer_Sandbox/Image_2/1'>alpha-carbon backbone representation</scene>. We colored the seven trans-membrane alpha helices green --- connected by loops that we colored gray.
 
--- We then displayed four sidechains <scene name='Hoelzer_Sandbox/Spacefilled_4/7'>(Phe 193, Trp 286, Phe 289, and Phe 290)</scene> involved in the binding of a beta-blocker, and colored them blue.  
 
--- The beta-blocker, <scene name='Hoelzer_Sandbox/Spacefilled_4/6'>Carazolol</scene>, was then added in a ball-and-stick format, colored orange.   
 
--- The <scene name='Hoelzer_Sandbox/Spacefilled_4/8'>three cholesterol molecules </scene> resolved in this structure, bound to the outside surface of the protein, were added and displayed in a ball-and-stick format, colored red.   
 
--- Finally, <scene name='Hoelzer_Sandbox/Spacefilled_4/9'>the N-terminal end </scene>of the protein was colored blue, and <scene name='Hoelzer_Sandbox/Spacefilled_4/10'>the C-terminal end </scene>was colored magenta.
 
A ply file describing this final structure was exported from RP-RasMol and sent to the MSOE Center for BioMolecular Modeling, where it was constructed from plaster powder, using a color ZCorp 3D printer.
 
{{Clear}}
 
==<font color = 'blue'>References</font>==
 
:Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, and Stevens RC. (2007) High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein-Coupled Receptor. Science. 318: 1258-1265.
 
{{Clear}}
 
==<font color = 'blue'>Our Poster and Presentations</font>==
 
[[Image:Madison West SMART Team at ASBMB.jpg|Madison West SMART Team| 350px]]
[[Image:Madison West SMART Team at Scripps.jpg|Madison West SMART Team| 350px]]
[[Image:Madison West SMART Team Poster - 2008.jpg|Madison West SMART Team| 350px]]
 
{{Clear}}
 
We were able to present our project, poster, and our physical models at the 2008 ASBMB meeting in San Diego, CA.  The poster was presented at the Medical College of Wisconsin and at the Scripps Research Institute.  During our visit to Scripps, we met Art Olson and David Goodsell --- from the Molecular Graphics Laboratory at TSRI.
 
 
==<font color = 'red'>MSOE Center for BioMolecular Modeling and SMART Teams</font>==
[[Image:Center for BioMolecular Modeling Logo.jpg|left|200px]]
[[Image:Smart Teams photo 5.jpg|right|120px]]


<font color = 'red'>SMART  Teams (S</font>tudents <font color = 'red'>M</font>odeling <font color = 'red'>A</font> <font color = 'red'>R</font>esearch <font color = 'red'>T</font>opic) is a science outreach program developed by the MSOE Center for BioMolecular Modeling.  In this program, teams of high school students work with a local resarch lab to design and build a physical model of a protein that is being investigated by the lab.  The goal of the SMART Team program is to introduce students to the real world of science --- as it exists in a local research lab.  The development of this program was supported by grants from the NIH-NCRR SEPA program (Science Education Partnership Award) and an HHMI Precollege Science Education Award.  For more information about this program, visit the CBM web site at [http://www.rpc.msoe.edu/cbm www.rpc.msoe.edu/cbm] .
[http://en.wikipedia.org/wiki/Glycoside_hydrolase#Retaining_glycoside_hydrolases Retaining Glycoside Hydrolases]
===References===
<references/>

Revision as of 23:10, 15 November 2009

Hen Egg White (HEW) Lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to the active site, PDBid 1HEW

Drag the structure with the mouse to rotate

Lysozyme was the first enzyme whose X-ray structure was determined [1][2]. This shows Hen Egg White (HEW) lysozyme containing a trisaccharide of N-acetylglucosamine (NAG) bound to a cleft in the enzyme. David Phillips, who determined the structure in 1965, saw that the cleft was large enough to fit three more saccharide units. He therefore built a model extending the trisaccharide to a that fits into the cleft, labeling the sugar subsites A-F[3]. Alternately click on and to turn the modeled portion of the hexasaccharide on and off. The interesting thing about the model was that the only way that the hexasaccharide would fit into the cleft was if the 4th saccharide (in subsite D) was strained into a . This conformation is what would be necessary for the formation of an oxocarbenium ion (oxionium ion). When the model was studied, was found to be in an ideal location to act as a general acid catalyst, 3.34 Angstroms from the bridging oxygen between the 4th and 5th saccharide units. appeared to be too far away (2.69 angstroms) in the static lysozyme structure to have formed a covalent bond with C1 of the half-chair model in the D site, and no covalent intermediate had ever been detected, so Phillips proposed that it acted as an electrostatic stabilizer of the oxonium ion (referred to as The Phillips Mechanism).

NAG-2-deoxy-2-fluoro-glucosyl fluoride (NAG2FGlcF) bound to Glu35Gln HEW Lysozyme PDBid 1H6M

Drag the structure with the mouse to rotate

Then, in 2001, Stephen Withers published ,[4] in which Glu 35 he had mutated to Gln to remove the general acid catalyst. The substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF). The fluoro group on C-1 does not require acid catalysis to be a good leaving group, and the remaining saccharide, in the absence of the acid necessary to catalyse the second step of the reaction, was demonstrated to form a . In this of the half chair model with 1HEW (greens) and the covalent intermediate in 1H6M (blues), note the relatively small motions of Asp 52 and C1 of the sugar ring in going from the model to the covalent intermediate. to observe the motion from the to the just toggle between the two green links.

Some Useful External LinksSome Useful External Links

Lysozyme

Retaining Glycoside Hydrolases

ReferencesReferences

  1. ↑ Johnson LN, Phillips DC. Structure of some crystalline lysozyme-inhibitor complexes determined by X-ray analysis at 6 Angstrom resolution. Nature. 1965 May 22;206(986):761-3. PMID:5840126
  2. ↑ Phillips, D. C. The hen egg white lysozyme molecule. Proc. Natl Acad. Sci. USA 57, 483-495 (1967)
  3. ↑ coordinates of the model kindly provided by Louise Johnson
  4. ↑ Vocadlo DJ, Davies GJ, Laine R, Withers SG. Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature. 2001 Aug 23;412(6849):835-8. PMID:11518970 doi:10.1038/35090602

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Joel L. Sussman
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