Functional effects of exchanging domains and linkers in transcription regulators

转录调节因子中交换域和连接子的功能效应

• 批准号: 8122158
• 负责人: LISKIN SWINT-KRUSE
• 金额: $ 25.93万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8122158
• 关键词: Address; Affect; Affinity; Agreement; Algorithms; Amino Acid Substitution; Amino Acids; Binding; Binding Sites; Bioinformatics; Biological Assay; Cartoons; Chimera organism; Chimeric Proteins; Communication; Complication; DNA; DNA Binding; DNA Binding Domain; Data; Data Set; Databases; Engineering; Escherichia coli; Family; Family Leave; Family member; Genetic Recombination; Genetic Transcription; Genomics; Goals; Gray unit of radiation dose; Homologous Gene; Human Genome Project; In Vitro; Knowledge; Left; Ligand Binding; Ligands; Link; Location; Measurement; Measures; Mediating; Monitor; Mutagenesis; N-terminal; Oranges; Outcome; Positioning Attribute; Protein Binding; Protein Region; Proteins; Relative (related person); Repression; Research Personnel; Role; Screening procedure; Sequence Analysis; Specificity; Structure; Tertiary Protein Structure; Testing; Thermodynamics; Transcriptional Regulation; Transplantation; Tweens; Variant; Work; design; dimer; functional gain; gene repression; improved; in vivo; multidisciplinary; novel; pathogen; programs; research study; response

DESCRIPTION (provided by applicant): Protein homologues often have common functions, with variation evolving via amino acid substitutions at specificity determinants (SDs). These residues may be located in binding sites or other "long-range" regions of the protein. Predicting SDs is a current target of bioinformatics sequence analyses. Our long- term goal is to experimentally identify long-range SDs in the Lacl/GaIR family; the proposed work focuses on SDs in the linker that connects the DMA-binding domain to the regulatory domain. Hypotheses are derived from 4 different prediction strategies, which are only in partial agreement with each other. The common function of the Lacl/GaIR proteins - transcription control - allows "moderate through-put" assays of function so that all predictions may be compared. We will test hypotheses in multiple homologues to address the question: Do homologues utilize all SDs available to the common fold (a frequent assumption of prediction algorithms) or do different functions require only a subset of potential SDs? Further, the aspect of function affected by changing an SD cannot yet be reliably predicted, nor is it clear whether a functional change for one SD is the same in all homologues. Our experiments will monitor different aspects of Lacl/GaIR function, including DMA specificity, DNA affinity, and allosteric response to binding regulatory effector molecules. Proposed experiments utilize chimeras comprising the Lacl DMA-binding domain and regulatory domains from E. coli paralogues. Linkers come from Lacl or paralogues. Since each naturally-occurring Lacl/GaIR protein recognizes a different DNA ligand, the common DNA-binding domain allows us to more easily parse functional contributions from binding site and long-range SDs. By definition, making an amino acid substitu- tion at an SD will change function. We will use in vivo repression/response to effector and in vitro thermody- namic measurements of affinity/allosteric response to characterize the chimeras and potential SD variants. Specificity for alternative DNA ligands will be determined. Experiments are designed to answer the following questions: Aim 1: Can one linker facilitate allosteric communication with a variety of regulatory domains? [or do altered linker SD interactions abolish this function?] Aim 2: What are the functional contributions from specific positions in the linker? Aim 3: Can knowledge of SDs [in one linker] be used to transplant lacO1 DNA-binding to other linkers? Results will: (1) Identify the locations of SDs and determine if they make similar functional contributions to several homologues; (2) Yield a list of empirical rules for creating novel Lacl/GaIR proteins with biotechnological utility via domain recombination; and (3) Test the current prediction algorithms and generate a new sequence/function database for improving predictions. This work will facilitate expanded use of data generated by the Human Genome Project.

DESCRIPTION (provided by applicant): Protein homologues often have common functions, with variation evolving via amino acid substitutions at specificity determinants (SDs). These residues may be located in binding sites or other "long-range" regions of the protein. Predicting SDs is a current target of bioinformatics sequence analyses. Our long- term goal is to experimentally identify long-range SDs in the Lacl/GaIR family; the proposed work focuses on SDs in the linker that connects the DMA-binding domain to the regulatory domain. Hypotheses are derived from 4 different prediction strategies, which are only in partial agreement with each other. The common function of the Lacl/GaIR proteins - transcription control - allows "moderate through-put" assays of function so that all predictions may be compared. We will test hypotheses in multiple homologues to address the question: Do homologues utilize all SDs available to the common fold (a frequent assumption of prediction algorithms) or do different functions require only a subset of potential SDs? Further, the aspect of function affected by changing an SD cannot yet be reliably predicted, nor is it clear whether a functional change for one SD is the same in all homologues. Our experiments will monitor different aspects of Lacl/GaIR function, including DMA specificity, DNA affinity, and allosteric response to binding regulatory effector molecules. Proposed experiments utilize chimeras comprising the Lacl DMA-binding domain and regulatory domains from E. coli paralogues. Linkers come from Lacl or paralogues. Since each naturally-occurring Lacl/GaIR protein recognizes a different DNA ligand, the common DNA-binding domain allows us to more easily parse functional contributions from binding site and long-range SDs. By definition, making an amino acid substitu- tion at an SD will change function. We will use in vivo repression/response to effector and in vitro thermody- namic measurements of affinity/allosteric response to characterize the chimeras and potential SD variants. Specificity for alternative DNA ligands will be determined. Experiments are designed to answer the following questions: Aim 1: Can one linker facilitate allosteric communication with a variety of regulatory domains? [or do altered linker SD interactions abolish this function?] Aim 2: What are the functional contributions from specific positions in the linker? Aim 3: Can knowledge of SDs [in one linker] be used to transplant lacO1 DNA-binding to other linkers? Results will: (1) Identify the locations of SDs and determine if they make similar functional contributions to several homologues; (2) Yield a list of empirical rules for creating novel Lacl/GaIR proteins with biotechnological utility via domain recombination; and (3) Test the current prediction algorithms and generate a new sequence/function database for improving predictions. This work will facilitate expanded use of data generated by the Human Genome Project.

项目成果

Data on publications, structural analyses, and queries used to build and utilize the AlloRep database.

用于构建和利用 AlloRep 数据库的出版物、结构分析和查询的数据。

• DOI: 10.1016/j.dib.2016.07.006
• 发表时间: 2016
• 期刊: Data in brief
• 影响因子: 1.2
• 作者: Sousa,FilipaL;Parente,DanielJ;Hessman,JacobA;Chazelle,Allen;Teichmann,SarahA;Swint-Kruse,Liskin
• 通讯作者: Swint-Kruse,Liskin

Multiple co-evolutionary networks are supported by the common tertiary scaffold of the LacI/GalR proteins.

• DOI: 10.1371/journal.pone.0084398
• 发表时间: 2013
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Parente DJ;Swint-Kruse L
• 通讯作者: Swint-Kruse L

Functionally important positions can comprise the majority of a protein's architecture.

功能上重要的位置可以构成蛋白质结构的大部分。

• DOI: 10.1002/prot.22985
• 发表时间: 2011
• 期刊: Proteins
• 影响因子: 2.9
• 作者: Tungtur,Sudheer;Parente,DanielJ;Swint-Kruse,Liskin
• 通讯作者: Swint-Kruse,Liskin

Modular, multi-input transcriptional logic gating with orthogonal LacI/GalR family chimeras.

• DOI: 10.1021/sb500262f
• 发表时间: 2014-09-19
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Shis, David L.;Hussain, Faiza;Meinhardt, Sarah;Swint-Kruse, Liskin;Bennett, Matthew R.
• 通讯作者: Bennett, Matthew R.

AlloRep: A Repository of Sequence, Structural and Mutagenesis Data for the LacI/GalR Transcription Regulators.

AlloRep：LacI/GalR 转录调节因子的序列、结构和诱变数据存储库。

• DOI: 10.1016/j.jmb.2015.09.015
• 发表时间: 2016
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Sousa,FilipaL;Parente,DanielJ;Shis,DavidL;Hessman,JacobA;Chazelle,Allen;Bennett,MatthewR;Teichmann,SarahA;Swint-Kruse,Liskin
• 通讯作者: Swint-Kruse,Liskin