A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding
The recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase high-performance liqu...
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| Veröffentlicht in: | Protein science 1999-11, Vol.8 (11), p.2513-2518 |
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| creator | DE YOUNG, LINDA R. SCHMELZER, CHARLES H. BURTON, LOUIS E. |
| description | The recombinant human nerve growth factor (hNGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF)
dimers all undergo rapid unfolding and dissociation to
monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase
high-performance liquid chromatography, and size-exclusion
chromatography were used to show that this monomer M1
converts slowly to a more fully unfolded monomer, M2,
by a first order process with half-lives of 22, 2.5, 1.6,
and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively,
at 25 °C. Linear Arrhenius plots for the conversion
of M1 to M2 yielded activation energies
of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and
BDNF, respectively. The refolding of these neurotrophins
from 5 M GdnHCl was also first order with NT-3 the slowest
to refold and BDNF the fastest. Threading of the N-terminus
out through the cystine-knot loop present in each of these
proteins is proposed as the slow step in unfolding. The
number of amino acids in the cystine-knot loop (14 for
hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number
and position of the proline residues in this loop (2 for
hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with
the relative rates of unfolding. The smaller the loop and
the greater the number of prolines, the more hindered and
slower the unfolding. |
| doi_str_mv | 10.1110/ps.8.11.2513 |
| format | Article |
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brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF)
dimers all undergo rapid unfolding and dissociation to
monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase
high-performance liquid chromatography, and size-exclusion
chromatography were used to show that this monomer M1
converts slowly to a more fully unfolded monomer, M2,
by a first order process with half-lives of 22, 2.5, 1.6,
and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively,
at 25 °C. Linear Arrhenius plots for the conversion
of M1 to M2 yielded activation energies
of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and
BDNF, respectively. The refolding of these neurotrophins
from 5 M GdnHCl was also first order with NT-3 the slowest
to refold and BDNF the fastest. Threading of the N-terminus
out through the cystine-knot loop present in each of these
proteins is proposed as the slow step in unfolding. The
number of amino acids in the cystine-knot loop (14 for
hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number
and position of the proline residues in this loop (2 for
hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with
the relative rates of unfolding. The smaller the loop and
the greater the number of prolines, the more hindered and
slower the unfolding.</description><identifier>ISSN: 0961-8368</identifier><identifier>EISSN: 1469-896X</identifier><identifier>DOI: 10.1110/ps.8.11.2513</identifier><identifier>PMID: 10595557</identifier><language>eng</language><publisher>Bristol: Cambridge University Press</publisher><subject>Amino Acid Sequence ; Animals ; Brain-Derived Neurotrophic Factor - chemistry ; Brain-Derived Neurotrophic Factor - metabolism ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; cystine‐knot ; Guanidine ; guanidine hydrochloride ; Humans ; Kinetics ; Macromolecular Substances ; Mice ; Models, Molecular ; Molecular Sequence Data ; nerve growth factor ; Nerve Growth Factors - chemistry ; Nerve Growth Factors - metabolism ; neurotrophic factor ; neurotrophin ; Neurotrophin 3 - chemistry ; Neurotrophin 3 - metabolism ; Protein Conformation ; Protein Denaturation ; protein folding ; Recombinant Proteins - chemistry ; Recombinant Proteins - metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; slow unfolding ; Thermodynamics</subject><ispartof>Protein science, 1999-11, Vol.8 (11), p.2513-2518</ispartof><rights>1999 The Protein Society</rights><rights>Copyright © 1999 The Protein Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4643-db16df272ccbd256cbdda9573e01b6359c139dd60cca76d667a096289fedd85b3</citedby><cites>FETCH-LOGICAL-c4643-db16df272ccbd256cbdda9573e01b6359c139dd60cca76d667a096289fedd85b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2144210/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2144210/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,1411,1427,27901,27902,45550,45551,46384,46808,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10595557$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>DE YOUNG, LINDA R.</creatorcontrib><creatorcontrib>SCHMELZER, CHARLES H.</creatorcontrib><creatorcontrib>BURTON, LOUIS E.</creatorcontrib><title>A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding</title><title>Protein science</title><addtitle>Protein Sci</addtitle><description>The recombinant human nerve growth factor (hNGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF)
dimers all undergo rapid unfolding and dissociation to
monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase
high-performance liquid chromatography, and size-exclusion
chromatography were used to show that this monomer M1
converts slowly to a more fully unfolded monomer, M2,
by a first order process with half-lives of 22, 2.5, 1.6,
and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively,
at 25 °C. Linear Arrhenius plots for the conversion
of M1 to M2 yielded activation energies
of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and
BDNF, respectively. The refolding of these neurotrophins
from 5 M GdnHCl was also first order with NT-3 the slowest
to refold and BDNF the fastest. Threading of the N-terminus
out through the cystine-knot loop present in each of these
proteins is proposed as the slow step in unfolding. The
number of amino acids in the cystine-knot loop (14 for
hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number
and position of the proline residues in this loop (2 for
hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with
the relative rates of unfolding. The smaller the loop and
the greater the number of prolines, the more hindered and
slower the unfolding.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Brain-Derived Neurotrophic Factor - chemistry</subject><subject>Brain-Derived Neurotrophic Factor - metabolism</subject><subject>Chromatography, Gel</subject><subject>Chromatography, High Pressure Liquid</subject><subject>cystine‐knot</subject><subject>Guanidine</subject><subject>guanidine hydrochloride</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Macromolecular Substances</subject><subject>Mice</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>nerve growth factor</subject><subject>Nerve Growth Factors - chemistry</subject><subject>Nerve Growth Factors - metabolism</subject><subject>neurotrophic factor</subject><subject>neurotrophin</subject><subject>Neurotrophin 3 - chemistry</subject><subject>Neurotrophin 3 - metabolism</subject><subject>Protein Conformation</subject><subject>Protein Denaturation</subject><subject>protein folding</subject><subject>Recombinant Proteins - chemistry</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Alignment</subject><subject>Sequence Homology, Amino Acid</subject><subject>slow unfolding</subject><subject>Thermodynamics</subject><issn>0961-8368</issn><issn>1469-896X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kU1PGzEQhi1UVALtrefKp56yqT_W3vWlEh8FKqGAKqpys7y2NzFa28HOFvHvcZQIUQlxmRl5Hr1-ZwaALxjNMMbo-yrP2lLNCMN0D0xwzUXVCn73AUyQ4LhqKW8PwGHO9wihGhP6ERxgxARjrJmAv8dQR-9jgN7qpQoue9jHBJMtz50LKqzhcvQqwPnF-RSenM1LnN9WdApVMNCPyQW76cE8xEc4hj4OxoXFJ7DfqyHbz7t8BP6c_7w9vayuri9-nR5fVbrmNa1Mh7npSUO07gxhvESjBGuoRbjjlAmNqTCGI61Vww3njSpDkVb01piWdfQI_NjqrsbOW6NtWCc1yFVyXqUnGZWT_3eCW8pF_CcJrmuCURH4thNI8WG0eS29y9oOgwo2jllyQWtGBCngdAvqFHNOtn_5BCO5uYRcZdmWSm4uUfCvr429grerL0C9BR7dYJ_eFZM3v69bjHe6s50N5bvkzMLK-zimUJb8tpFnX7ui-Q</recordid><startdate>19991101</startdate><enddate>19991101</enddate><creator>DE YOUNG, LINDA R.</creator><creator>SCHMELZER, CHARLES H.</creator><creator>BURTON, LOUIS E.</creator><general>Cambridge University Press</general><general>Cold Spring Harbor Laboratory Press</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>7X8</scope><scope>5PM</scope></search><sort><creationdate>19991101</creationdate><title>A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding</title><author>DE YOUNG, LINDA R. ; SCHMELZER, CHARLES H. ; BURTON, LOUIS E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4643-db16df272ccbd256cbdda9573e01b6359c139dd60cca76d667a096289fedd85b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Brain-Derived Neurotrophic Factor - chemistry</topic><topic>Brain-Derived Neurotrophic Factor - metabolism</topic><topic>Chromatography, Gel</topic><topic>Chromatography, High Pressure Liquid</topic><topic>cystine‐knot</topic><topic>Guanidine</topic><topic>guanidine hydrochloride</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Macromolecular Substances</topic><topic>Mice</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>nerve growth factor</topic><topic>Nerve Growth Factors - chemistry</topic><topic>Nerve Growth Factors - metabolism</topic><topic>neurotrophic factor</topic><topic>neurotrophin</topic><topic>Neurotrophin 3 - chemistry</topic><topic>Neurotrophin 3 - metabolism</topic><topic>Protein Conformation</topic><topic>Protein Denaturation</topic><topic>protein folding</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Alignment</topic><topic>Sequence Homology, Amino Acid</topic><topic>slow unfolding</topic><topic>Thermodynamics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>DE YOUNG, LINDA R.</creatorcontrib><creatorcontrib>SCHMELZER, CHARLES H.</creatorcontrib><creatorcontrib>BURTON, LOUIS E.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Protein science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>DE YOUNG, LINDA R.</au><au>SCHMELZER, CHARLES H.</au><au>BURTON, LOUIS E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding</atitle><jtitle>Protein science</jtitle><addtitle>Protein Sci</addtitle><date>1999-11-01</date><risdate>1999</risdate><volume>8</volume><issue>11</issue><spage>2513</spage><epage>2518</epage><pages>2513-2518</pages><issn>0961-8368</issn><eissn>1469-896X</eissn><abstract>The recombinant human nerve growth factor (hNGF),
brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF)
dimers all undergo rapid unfolding and dissociation to
monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase
high-performance liquid chromatography, and size-exclusion
chromatography were used to show that this monomer M1
converts slowly to a more fully unfolded monomer, M2,
by a first order process with half-lives of 22, 2.5, 1.6,
and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively,
at 25 °C. Linear Arrhenius plots for the conversion
of M1 to M2 yielded activation energies
of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and
BDNF, respectively. The refolding of these neurotrophins
from 5 M GdnHCl was also first order with NT-3 the slowest
to refold and BDNF the fastest. Threading of the N-terminus
out through the cystine-knot loop present in each of these
proteins is proposed as the slow step in unfolding. The
number of amino acids in the cystine-knot loop (14 for
hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number
and position of the proline residues in this loop (2 for
hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with
the relative rates of unfolding. The smaller the loop and
the greater the number of prolines, the more hindered and
slower the unfolding.</abstract><cop>Bristol</cop><pub>Cambridge University Press</pub><pmid>10595557</pmid><doi>10.1110/ps.8.11.2513</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0961-8368 |
| ispartof | Protein science, 1999-11, Vol.8 (11), p.2513-2518 |
| issn | 0961-8368 1469-896X |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2144210 |
| source | Wiley Free Content; MEDLINE; Wiley Online Library Journals Frontfile Complete; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence Animals Brain-Derived Neurotrophic Factor - chemistry Brain-Derived Neurotrophic Factor - metabolism Chromatography, Gel Chromatography, High Pressure Liquid cystine‐knot Guanidine guanidine hydrochloride Humans Kinetics Macromolecular Substances Mice Models, Molecular Molecular Sequence Data nerve growth factor Nerve Growth Factors - chemistry Nerve Growth Factors - metabolism neurotrophic factor neurotrophin Neurotrophin 3 - chemistry Neurotrophin 3 - metabolism Protein Conformation Protein Denaturation protein folding Recombinant Proteins - chemistry Recombinant Proteins - metabolism Sequence Alignment Sequence Homology, Amino Acid slow unfolding Thermodynamics |
| title | A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding |
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