Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS
Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as...
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Veröffentlicht in: | J. Am. Soc. Mass. Spectrom 2012-03, Vol.23 (3), p.505-519 |
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creator | Sides, Cynthia R. Liyanage, Rohana Lay, Jackson O. Philominathan, Sagaya Theresa Leena Matsushita, Osamu Sakon, Joshua |
description | Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound. |
doi_str_mv | 10.1007/s13361-011-0309-3 |
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(ANL), Argonne, IL (United States). Advanced Photon Source (APS)</creatorcontrib><description>Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.</description><identifier>ISSN: 1044-0305</identifier><identifier>EISSN: 1879-1123</identifier><identifier>DOI: 10.1007/s13361-011-0309-3</identifier><identifier>PMID: 22207568</identifier><language>eng</language><publisher>New York: Springer-Verlag</publisher><subject>Accessibility ; Analytical Chemistry ; Apoenzymes - chemistry ; Apoenzymes - metabolism ; Bacteria ; Binding ; Binding Sites ; Bioinformatics ; Biotechnology ; Calcium ; Calcium - chemistry ; Chemistry ; Chemistry and Materials Science ; Cleavage ; Clostridium histolyticum - enzymology ; Cobalt ; Cobalt - chemistry ; Crystal structure ; Crystallography ; Crystallography, X-Ray ; Desorption ; Drug delivery systems ; Dynamic stability ; Enzyme Stability ; Ionization ; Ions ; Lasers ; Mass spectrometry ; Microbial Collagenase - chemistry ; Microbial Collagenase - metabolism ; Models, Molecular ; Organic Chemistry ; Peptide Fragments - chemistry ; Peptide Fragments - metabolism ; Phase stability ; Protein Conformation ; Proteins ; Proteomics ; Research Article ; Serine Endopeptidases - chemistry ; Serine Endopeptidases - metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods ; Stability analysis ; Structural stability ; Trypsin - chemistry ; Trypsin - metabolism ; X-ray scattering</subject><ispartof>J. 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(ANL), Argonne, IL (United States). Advanced Photon Source (APS)</creatorcontrib><title>Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS</title><title>J. Am. Soc. Mass. Spectrom</title><addtitle>J. Am. Soc. Mass Spectrom</addtitle><addtitle>J Am Soc Mass Spectrom</addtitle><description>Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.</description><subject>Accessibility</subject><subject>Analytical Chemistry</subject><subject>Apoenzymes - chemistry</subject><subject>Apoenzymes - metabolism</subject><subject>Bacteria</subject><subject>Binding</subject><subject>Binding Sites</subject><subject>Bioinformatics</subject><subject>Biotechnology</subject><subject>Calcium</subject><subject>Calcium - chemistry</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Cleavage</subject><subject>Clostridium histolyticum - enzymology</subject><subject>Cobalt</subject><subject>Cobalt - chemistry</subject><subject>Crystal structure</subject><subject>Crystallography</subject><subject>Crystallography, X-Ray</subject><subject>Desorption</subject><subject>Drug delivery systems</subject><subject>Dynamic stability</subject><subject>Enzyme Stability</subject><subject>Ionization</subject><subject>Ions</subject><subject>Lasers</subject><subject>Mass spectrometry</subject><subject>Microbial Collagenase - chemistry</subject><subject>Microbial Collagenase - metabolism</subject><subject>Models, Molecular</subject><subject>Organic Chemistry</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - metabolism</subject><subject>Phase stability</subject><subject>Protein Conformation</subject><subject>Proteins</subject><subject>Proteomics</subject><subject>Research Article</subject><subject>Serine Endopeptidases - chemistry</subject><subject>Serine Endopeptidases - metabolism</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods</subject><subject>Stability analysis</subject><subject>Structural stability</subject><subject>Trypsin - chemistry</subject><subject>Trypsin - metabolism</subject><subject>X-ray scattering</subject><issn>1044-0305</issn><issn>1879-1123</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNp1Ul1rFDEUHUSxdfUH-CJBH1ToaG4ymY8Xod21WthSofU5ZDJ3dlNmkm2SKexv8c-aZetSBR9CLpyTc8-9OVn2GugnoLT6HIDzEnIK6XDa5PxJdgx11eQAjD9NNS2KHSKOshch3FIKFW2q59kRY4xWoqyPs18_vGuNXZG4RsLzBbmOftJx8nhCFlurRqPDCVG2S4BqzWDilrienCkd0Rs1kLkbBrVCqwIeanJmbLcTXbhRGUs-qI3LyT36MAWydoPLP5J2S5ZmNBE7kixEdMM2mEAuT5eLi_zm6pxcXr_MnvVqCPjq4Z5lP8-_3sy_58urbxfz02WuRclirsoWhODQgQJo61KVrEQFbVEp4L0WUHe6VJXqGOq-1kVZ0qavQCD0LVM98ln2Za-7mdoRO402ejXIjTej8lvplJF_I9as5crdS87rhguWBN7uBVyIRgadptJr7axFHSXQuhZFkUjvH7p4dzdhiHI0QWPamEU3BdkwVommgh3z3T_MWzd5m1YgoRG0aVj60sSCPUt7F4LH_mAYqNzFQ-7jIVM85C4eye4se_N40sOLP3lIBLYnhATZFfpHrf-r-hvaBcVQ</recordid><startdate>20120301</startdate><enddate>20120301</enddate><creator>Sides, Cynthia R.</creator><creator>Liyanage, Rohana</creator><creator>Lay, Jackson O.</creator><creator>Philominathan, Sagaya Theresa Leena</creator><creator>Matsushita, Osamu</creator><creator>Sakon, Joshua</creator><general>Springer-Verlag</general><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FG</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>P5Z</scope><scope>P62</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>OTOTI</scope><scope>5PM</scope></search><sort><creationdate>20120301</creationdate><title>Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS</title><author>Sides, Cynthia R. ; 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(ANL), Argonne, IL (United States). 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Am. Soc. Mass. Spectrom</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sides, Cynthia R.</au><au>Liyanage, Rohana</au><au>Lay, Jackson O.</au><au>Philominathan, Sagaya Theresa Leena</au><au>Matsushita, Osamu</au><au>Sakon, Joshua</au><aucorp>Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS</atitle><jtitle>J. Am. Soc. Mass. Spectrom</jtitle><stitle>J. Am. Soc. Mass Spectrom</stitle><addtitle>J Am Soc Mass Spectrom</addtitle><date>2012-03-01</date><risdate>2012</risdate><volume>23</volume><issue>3</issue><spage>505</spage><epage>519</epage><pages>505-519</pages><issn>1044-0305</issn><eissn>1879-1123</eissn><abstract>Pairing limited proteolysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to probe clostridial collagenase collagen binding domain (CBD) reveals the solution dynamics and stability of the protein, as these factors are crucial to CBD effectiveness as a drug-delivery vehicle. MS analysis of proteolytic digests indicates initial cleavage sites, thereby specifying the less stable and highly accessible regions of CBD. Modulation of protein structure and stability upon metal binding is shown through MS analysis of calcium-bound and cobalt-bound CBD proteolytic digests. Previously determined X-ray crystal structures illustrate that calcium binding induces secondary structure transformation in the highly mobile N-terminal arm and increases protein stability. MS-based detection of exposed residues confirms protein flexibility, accentuates N-terminal dynamics, and demonstrates increased global protein stability exported by calcium binding. Additionally, apo- and calcium-bound CBD proteolysis sites correlate well with crystallographic B-factors, accessibility, and enzyme specificity. MS-observed cleavage sites with no clear correlations are explained either by crystal contacts of the X-ray crystal structures or by observed differences between Molecules A and B in the X-ray crystal structures. The study newly reveals the absence of the βA strand and thus the very dynamic N-terminal linker, as corroborated by the solution X-ray scattering results. Cobalt binding has a regional effect on the solution phase stability of CBD, as limited proteolysis data implies the capture of an intermediate-CBD solution structure when cobalt is bound.</abstract><cop>New York</cop><pub>Springer-Verlag</pub><pmid>22207568</pmid><doi>10.1007/s13361-011-0309-3</doi><tpages>15</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Accessibility Analytical Chemistry Apoenzymes - chemistry Apoenzymes - metabolism Bacteria Binding Binding Sites Bioinformatics Biotechnology Calcium Calcium - chemistry Chemistry Chemistry and Materials Science Cleavage Clostridium histolyticum - enzymology Cobalt Cobalt - chemistry Crystal structure Crystallography Crystallography, X-Ray Desorption Drug delivery systems Dynamic stability Enzyme Stability Ionization Ions Lasers Mass spectrometry Microbial Collagenase - chemistry Microbial Collagenase - metabolism Models, Molecular Organic Chemistry Peptide Fragments - chemistry Peptide Fragments - metabolism Phase stability Protein Conformation Proteins Proteomics Research Article Serine Endopeptidases - chemistry Serine Endopeptidases - metabolism Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods Stability analysis Structural stability Trypsin - chemistry Trypsin - metabolism X-ray scattering |
title | Probing the 3-D Structure, Dynamics, and Stability of Bacterial Collagenase Collagen Binding Domain (apo- versus holo-) by Limited Proteolysis MALDI-TOF MS |
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