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1.
J Vis Exp ; (207)2024 May 31.
Article in English | MEDLINE | ID: mdl-38884467

ABSTRACT

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.


Subject(s)
Quorum Sensing , Trans-Activators , Vibrio , Quorum Sensing/drug effects , Vibrio/drug effects , Vibrio/chemistry , Vibrio/metabolism , Vibrio/genetics , Trans-Activators/antagonists & inhibitors , Trans-Activators/genetics , Trans-Activators/metabolism , Trans-Activators/chemistry , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Repressor Proteins/metabolism , Repressor Proteins/chemistry , Sulfonamides/pharmacology , Sulfonamides/chemistry , Thiophenes/chemistry , Thiophenes/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry
2.
Mol Microbiol ; 116(4): 1173-1188, 2021 10.
Article in English | MEDLINE | ID: mdl-34468051

ABSTRACT

The quorum-sensing signaling systems in Vibrio bacteria converge to control levels of the master transcription factors LuxR/HapR, a family of highly conserved proteins that regulate gene expression for bacterial behaviors. A compound library screen identified 2-thiophenesulfonamide compounds that specifically inhibit Vibrio campbellii LuxR but do not affect cell growth. We synthesized a panel of 50 thiophenesulfonamide compounds to examine the structure-activity relationship effects on Vibrio quorum sensing. The most potent molecule identified, PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole), inhibits quorum sensing in multiple strains of V. vulnificus, V. parahaemolyticus, and V. campbellii at nanomolar concentrations. However, thiophenesulfonamide inhibition efficacy varies significantly among Vibrio species: PTSP is most inhibitory against V. vulnificus SmcR, but V. cholerae HapR is completely resistant to all thiophenesulfonamides tested. Reverse genetics experiments show that PTSP efficacy is dictated by amino acid sequence in the putative ligand-binding pocket: F75Y and C170F SmcR substitutions are each sufficient to eliminate PTSP inhibition. Further, in silico modeling distinguished the most potent thiophenesulfonamides from less-effective derivatives. Our results revealed the previously unknown differences in LuxR/HapR proteins that control quorum sensing in Vibrio species and underscore the potential for developing thiophenesulfonamides as specific quorum sensing-directed treatments for Vibrio infections.


Subject(s)
Quorum Sensing/drug effects , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Sulfonamides/metabolism , Sulfonamides/pharmacology , Trans-Activators/antagonists & inhibitors , Trans-Activators/metabolism , Vibrio/metabolism , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Ligands , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Repressor Proteins/chemistry , Species Specificity , Structure-Activity Relationship , Sulfonamides/chemistry , Trans-Activators/chemistry , Vibrio/chemistry , Vibrio/genetics
3.
J Phys Chem B ; 123(40): 8387-8396, 2019 10 10.
Article in English | MEDLINE | ID: mdl-31535866

ABSTRACT

Flexible protein sequences populate ensembles of rapidly interconverting states differentiated by small-scale fluctuations; however, elucidating whether and how the ensembles determine function experimentally is challenged by the combined high spatial and temporal resolution needed to capture the states. We used carbon-deuterium (C-D) bond vibrations incorporated as infrared probes to characterize with residue-specific detail the heterogeneity of states adopted by proline-rich (PR) sequences and assess their involvement in recognition of Src homology 3 domains. The C-D absorption envelopes provided evidence for two or three sub-populations at all proline residues. The changes in the subpopulations induced by binding generally reflected recognition by conformational selection but depended on the residue and the state of the ligand to illuminate distinct mechanisms among the PR ligands. Notably, the spectral data indicate that greater adaptability among the states is associated with reduced recognition specificity and that perturbation to the ensemble populations contributes to differences in binding entropy. Broadly, the study quantifies rapidly interconverting ensembles with residue-specific detail and implicates them in function.


Subject(s)
Molecular Dynamics Simulation , Proteins/chemistry , Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Entropy , Kinetics , Peptides/chemistry , Peptides/metabolism , src Homology Domains
4.
J Phys Chem B ; 123(9): 2114-2122, 2019 03 07.
Article in English | MEDLINE | ID: mdl-30742428

ABSTRACT

Transient protein complexes are crucial for sustaining dynamic cellular processes. The complexes of electron-transfer proteins are a notable example, such as those formed by plastocyanin (Pc) and cytochrome f (cyt f) in the photosynthetic apparatus. The dynamic and heterogeneous nature of these complexes, however, makes their study challenging. To better elucidate the complex of Nostoc Pc and cyt f, 2D-IR spectroscopy coupled to site-specific labeling with cyanophenylalanine infrared (IR) probes was employed to characterize how the local environments at sites along the surface of Pc were impacted by cyt f binding. The results indicate that Pc most substantially engages with cyt f via the hydrophobic patch around the copper redox site. Complexation with cyt f led to an increase in inhomogeneous broadening of the probe absorptions, reflective of increased heterogeneity of interactions with their environment. Notably, most of the underlying states interconverted very rapidly (1 to 2 ps), suggesting a complex with a highly mobile interface. The data support a model of the complex consisting of a large population of an encounter complex. Additionally, the study demonstrates the application of 2D-IR spectroscopy with site-specifically introduced probes to reveal new quantitative insight about dynamic biochemical systems.


Subject(s)
Cytochromes f/metabolism , Plastocyanin/metabolism , Alanine/analogs & derivatives , Alanine/chemistry , Binding Sites , Cytochromes f/chemistry , Hydrophobic and Hydrophilic Interactions , Molecular Probes/chemistry , Nitriles/chemistry , Nostoc/chemistry , Plastocyanin/chemistry , Protein Binding , Spectroscopy, Fourier Transform Infrared
5.
Phys Chem Chem Phys ; 21(2): 780-788, 2019 Jan 02.
Article in English | MEDLINE | ID: mdl-30548035

ABSTRACT

The conformational heterogeneity and dynamics of protein side chains contribute to function, but investigating exactly how is hindered by experimental challenges arising from the fast timescales involved and the spatial heterogeneity of protein structures. The potential of two-dimensional infrared (2D IR) spectroscopy for measuring conformational heterogeneity and dynamics with unprecedented spatial and temporal resolution has motivated extensive effort to develop amino acids with functional groups that have frequency-resolved absorptions to serve as probes of their protein microenvironments. We demonstrate the full advantage of the approach by selective incorporation of the probe p-cyanophenylalanine at six distinct sites in a Src homology 3 domain and the application of 2D IR spectroscopy to site-specifically characterize heterogeneity and dynamics and their contribution to cognate ligand binding. The approach revealed a wide range of microenvironments and distinct responses to ligand binding, including at the three adjacent, conserved aromatic residues that form the recognition surface of the protein. Molecular dynamics simulations performed for all the labeled proteins provide insight into the underlying heterogeneity and dynamics. Similar application of 2D IR spectroscopy and site-selective probe incorporation will allow for the characterization of heterogeneity and dynamics of other proteins, how heterogeneity and dynamics are affected by solvation and local structure, and how they might contribute to biological function.


Subject(s)
Chemistry Techniques, Analytical/instrumentation , Chemistry Techniques, Analytical/methods , Proteins/chemistry , Spectrophotometry, Infrared , Molecular Dynamics Simulation , src Homology Domains
6.
J Am Chem Soc ; 138(4): 1130-3, 2016 Feb 03.
Article in English | MEDLINE | ID: mdl-26784847

ABSTRACT

Conformational heterogeneity and dynamics are increasingly evoked in models of protein molecular recognition but are challenging to experimentally characterize. Here we combine the inherent temporal resolution of infrared (IR) spectroscopy with the spatial resolution afforded by selective incorporation of carbon-deuterium (C-D) bonds, which provide frequency-resolved absorptions within a protein IR spectrum, to characterize the molecular recognition of the Src homology 3 (SH3) domain of the yeast protein Sho1 with its cognate proline-rich (PR) sequence of Pbs2. The IR absorptions of C-D bonds introduced at residues along a peptide of the Pbs2 PR sequence report on the changes in the local environments upon binding to the SH3 domain. Interestingly, upon forming the complex the IR spectra of the peptides labeled with C-D bonds at either of the two conserved prolines of the PXXP consensus recognition sequence show more absorptions than there are C-D bonds, providing evidence for the population of multiple states. In contrast, the NMR spectra of the peptides labeled with (13)C at the same residues show only single resonances, indicating rapid interconversion on the NMR time scale. Thus, the data suggest that the SH3 domain recognizes its cognate peptide with a component of induced fit molecular recognition involving the adoption of multiples states, which have previously gone undetected due to interconversion between the populated states that is too fast to resolve using conventional methods.


Subject(s)
Proline/chemistry , Proteins/chemistry , src Homology Domains , Binding Sites , Carbon-13 Magnetic Resonance Spectroscopy , Protein Conformation , Spectroscopy, Fourier Transform Infrared
7.
Anal Methods ; 7: 7234-7241, 2015.
Article in English | MEDLINE | ID: mdl-26491469

ABSTRACT

Local heterogeneity of microenvironments in proteins is important in biological function, but difficult to characterize experimentally. One approach is the combination of infrared (IR) spectroscopy and site-selective incorporation of probe moieties with spectrally resolved IR absorptions that enable characterization within inherently congested protein IR spectra. We employed this method to study molecular recognition of a Src homology 3 (SH3) domain from the yeast protein Sho1 for a peptide containing the proline-rich recognition sequence of its physiological binding partner Pbs2. Nitrile IR probes were introduced at four distinct sites in the protein by selective incorporation of p-cyanophenylalanine via the amber codon suppressor method and characterized by IR spectroscopy. Variation among the IR absorption bands reports on heterogeneity in local residue environments dictated by the protein structure, as well as on residue-dependent changes upon peptide binding. The study informs on the molecular recognition of SH3 Sho1 and illustrates the speed and simplicity of this approach for characterization of select microenvironments within proteins.

8.
Analyst ; 140(13): 4336-49, 2015 Jul 07.
Article in English | MEDLINE | ID: mdl-26007625

ABSTRACT

Two-dimensional infrared (2D IR) spectroscopy has recently emerged as a powerful tool with applications in many areas of scientific research. The inherent high time resolution coupled with bond-specific spatial resolution of IR spectroscopy enable direct characterization of rapidly interconverting species and fast processes, even in complex systems found in chemistry and biology. In this minireview, we briefly outline the fundamental principles and experimental procedures of 2D IR spectroscopy. Using illustrative example studies, we explain the important features of 2D IR spectra and their capability to elucidate molecular structure and dynamics. Primarily, this minireview aims to convey the scope and potential of 2D IR spectroscopy by highlighting select examples of recent applications including the use of innate or introduced vibrational probes for the study of nucleic acids, peptides/proteins, and materials.


Subject(s)
Spectrophotometry, Infrared/methods , Animals , Humans , Nucleic Acids/analysis , Nucleic Acids/chemistry , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins/analysis , Proteins/chemistry , Spectrophotometry, Infrared/statistics & numerical data
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