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1.
Planta Med ; 80(8-9): 732-9, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24963620

ABSTRACT

A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R(2) = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Metabolomics , Plant Extracts/isolation & purification , Vaccinium/chemistry , Chlorogenic Acid/standards , Plant Extracts/chemistry , Plant Leaves/chemistry , Principal Component Analysis , Reference Standards , Species Specificity , Vaccinium/classification
2.
Biochemistry ; 52(52): 9510-8, 2013 Dec 31.
Article in English | MEDLINE | ID: mdl-24319994

ABSTRACT

Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.


Subject(s)
Eukaryotic Initiation Factor-1/metabolism , Peptide Initiation Factors/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Eukaryotic Initiation Factor-1/chemistry , Eukaryotic Initiation Factor-1/genetics , Humans , Models, Molecular , Molecular Sequence Data , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/genetics , Protein Binding , Protein Biosynthesis , Protein Multimerization , Protein Structure, Tertiary , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Eukaryotic Translation Initiation Factor 5A
3.
Cell Rep ; 1(6): 689-702, 2012 Jun 28.
Article in English | MEDLINE | ID: mdl-22813744

ABSTRACT

Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2ß on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2ß. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2ß in switching PICs from an open to a closed state at start codons.


Subject(s)
Codon, Initiator/metabolism , Eukaryotic Initiation Factor-1/metabolism , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-5/chemistry , Eukaryotic Initiation Factor-5/metabolism , Amino Acid Sequence , Amino Acid Substitution/genetics , Binding Sites , Conserved Sequence , Epitopes/metabolism , Eukaryotic Initiation Factor-1/chemistry , Eukaryotic Initiation Factor-2/chemistry , Evolution, Molecular , Gene Deletion , Genetic Complementation Test , Humans , Kinetics , Lysine/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Phenotype , Protein Binding , Protein Structure, Tertiary , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Scattering, Small Angle , Structure-Activity Relationship , X-Ray Diffraction
4.
J Org Chem ; 75(4): 1077-86, 2010 Feb 19.
Article in English | MEDLINE | ID: mdl-20095540

ABSTRACT

It has been widely accepted that both the protection of carbonyls and the deprotection of acetals and ketals involve the participation of a water molecule: formation of acetals and ketals is a dehydration process, whereas the deprotection is often referred to as hydrolysis, which, as implied by its name, always requires the presence of water. Herein, we report experimental evidence and mechanistic investigations that provide an alternative view to this process. We have demonstrated that water is not required to convert acetals and ketals to the corresponding carbonyls. The (1)H NMR experimental results revealed that the TFA-mediated transformation of acetal to aldehyde occurs via a hemiacetal TFA ester intermediate, which differentiates itself from the classic acid-catalyzed hydrolysis, where the hemiacetal is the putative intermediate responsible for the formation of the aldehyde. More interestingly, alcohols are not the final byproducts as they are in the classical hydrolysis, rather, the two alcohol molecules are converted to two TFA esters under the reaction conditions. On the basis of the NMR evidence, we have proposed that the two TFA esters are formed in two separate steps via a different mechanism along the reaction pathway. Formation of the TFA esters renders the reaction irreversible. To the best of our knowledge, the cascade reaction pathway presented by the TFA-mediated conversion of acetals and ketals to carbonyls has never been previously postulated.

6.
J Mol Biol ; 367(4): 1007-22, 2007 Apr 06.
Article in English | MEDLINE | ID: mdl-17292917

ABSTRACT

The L11 binding site is one of the most important functional sites in the ribosome. The N-terminal domain of L11 has been implicated as a "reversible switch" in facilitating the coordinated movements associated with EF-G-driven GTP hydrolysis. The reversible switch mechanism has been hypothesized to require conformational flexibility involving re-orientation and re-positioning of the two L11 domains, and warrants a close examination of the structure and dynamics of L11. Here we report the solution structure of free L11, and relaxation studies of free L11, L11 complexed to its 58 nt RNA recognition site, and L11 in a ternary complex with the RNA and thiostrepton antibiotic. The binding site of thiostrepton on L11 was also defined by analysis of structural and dynamics data and chemical shift mapping. The conclusions of this work are as follows: first, the binding of L11 to RNA leads to sizable conformation changes in the regions flanking the linker and in the hinge area that links a beta-sheet and a 3(10)-helix-turn-helix element in the N terminus. Concurrently, the change in the relative orientation may lead to re-positioning of the N terminus, as implied by a decrease of radius of gyration from 18.5 A to 16.2 A. Second, the regions, which undergo large conformation changes, exhibit motions on milliseconds-microseconds or nanoseconds-picoseconds time scales. Third, binding of thiostrepton results in more rigid conformations near the linker (Thr71) and near its putative binding site (Leu12). Lastly, conformational changes in the putative thiostrepton binding site are implicated by the re-emergence of cross-correlation peaks in the spectrum of the ternary complex, which were missing in that of the binary complex. Our combined analysis of both the chemical shift perturbation and dynamics data clearly indicates that thiostrepton binds to a pocket involving residues in the 3(10)-helix in L11.


Subject(s)
Ribosomal Proteins/chemistry , Thermus thermophilus/chemistry , Thiostrepton/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Crystallography, X-Ray , Models, Molecular , Multiprotein Complexes/chemistry , Protein Binding , Protein Conformation/drug effects , RNA, Bacterial/metabolism , Ribosomal Proteins/metabolism , Scattering, Small Angle , Thermus thermophilus/drug effects , Thiostrepton/metabolism , Thiostrepton/pharmacology
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