Role of a Surface Tryptophan in Defining the Structure, Stability, and DNA Binding of the Hyperthermophile Protein Sac7d
Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein−DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding...
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Veröffentlicht in: | Biochemistry (Easton) 2005-01, Vol.44 (3), p.915-925 |
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description | Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein−DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (ΔG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 °C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed. |
doi_str_mv | 10.1021/bi047823b |
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The crystal structure of the protein−DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (ΔG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 °C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. 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The crystal structure of the protein−DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (ΔG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 °C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed.</description><subject>Archaeal Proteins - metabolism</subject><subject>Calorimetry, Differential Scanning</subject><subject>Circular Dichroism</subject><subject>DNA - metabolism</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>Nuclear Magnetic Resonance, Biomolecular</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Spectrometry, Fluorescence</subject><subject>Spectrophotometry, Ultraviolet</subject><subject>Sulfolobus acidocaldarius</subject><subject>Surface Properties</subject><subject>Thermodynamics</subject><subject>Tryptophan - chemistry</subject><subject>Tryptophan - metabolism</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0U1r3DAQBmBRWppNmkP_QNGlhUDcjizJ8h7z1aSwpCG77VVI8rhR6rVdSYbsv4-WXdJLoSeN0MM7g4aQ9ww-MyjZF-tBqLrk9hWZMVlCIeZz-ZrMAKAqynkFB-Qwxsd8FaDEW3LAZCWFEmpGnu6HDunQUkOXU2iNQ7oKmzEN44Ppqe_pJba-9_0vmh6QLlOYXJoCnubSWN_5tDmlpm_o5e0ZPfd9s5U5bYtvNiOGXIR1DvO5y10YEubIpXGqeUfetKaLeLw_j8iPr1eri5ti8f3628XZojC8LlNRowODFQqL6BwvwVhrSglomWQIVSmNtKVr25qrmkFjDWtay7moFAjXNPyIfNrljmH4M2FMeu2jw64zPQ5T1JXiNaha_hcyVYMQSmV4soMuDDEGbPUY_NqEjWagt_vQL_vI9sM-dLJrbP7K_QIyKHbAx4RPL-8m_N5OpqRe3S319e1qwec_zzXP_uPOGxf14zCFPn_ePxo_A6EroT4</recordid><startdate>20050125</startdate><enddate>20050125</enddate><creator>Bedell, Jennifer L</creator><creator>Edmondson, Stephen P</creator><creator>Shriver, John W</creator><general>American Chemical Society</general><scope>BSCLL</scope><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>7TM</scope><scope>7X8</scope></search><sort><creationdate>20050125</creationdate><title>Role of a Surface Tryptophan in Defining the Structure, Stability, and DNA Binding of the Hyperthermophile Protein Sac7d</title><author>Bedell, Jennifer L ; Edmondson, Stephen P ; Shriver, John W</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a382t-8ec0ae6e4beecc320abba250eb151e0625a5b2cff837810dba1dfb3346704cdd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2005</creationdate><topic>Archaeal Proteins - metabolism</topic><topic>Calorimetry, Differential Scanning</topic><topic>Circular Dichroism</topic><topic>DNA - metabolism</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Nuclear Magnetic Resonance, Biomolecular</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Spectrometry, Fluorescence</topic><topic>Spectrophotometry, Ultraviolet</topic><topic>Sulfolobus acidocaldarius</topic><topic>Surface Properties</topic><topic>Thermodynamics</topic><topic>Tryptophan - chemistry</topic><topic>Tryptophan - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bedell, Jennifer L</creatorcontrib><creatorcontrib>Edmondson, Stephen P</creatorcontrib><creatorcontrib>Shriver, John W</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bedell, Jennifer L</au><au>Edmondson, Stephen P</au><au>Shriver, John W</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Role of a Surface Tryptophan in Defining the Structure, Stability, and DNA Binding of the Hyperthermophile Protein Sac7d</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2005-01-25</date><risdate>2005</risdate><volume>44</volume><issue>3</issue><spage>915</spage><epage>925</epage><pages>915-925</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein−DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (ΔG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 °C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>15654747</pmid><doi>10.1021/bi047823b</doi><tpages>11</tpages></addata></record> |
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subjects | Archaeal Proteins - metabolism Calorimetry, Differential Scanning Circular Dichroism DNA - metabolism DNA-Binding Proteins - metabolism Nuclear Magnetic Resonance, Biomolecular Protein Binding Protein Conformation Spectrometry, Fluorescence Spectrophotometry, Ultraviolet Sulfolobus acidocaldarius Surface Properties Thermodynamics Tryptophan - chemistry Tryptophan - metabolism |
title | Role of a Surface Tryptophan in Defining the Structure, Stability, and DNA Binding of the Hyperthermophile Protein Sac7d |
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