Use of 2-aminopurine as a fluorescent tool for characterizing antibiotic recognition of the bacterial rRNA A-site

Barbieri, Christopher M.; Kaul, Malvika; Pilch, Daniel S.

doi:10.1016/j.tet.2006.08.107

Cited by 35 publications

(40 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fluorescence spectra were obtained using a spectrofluorimeter equipped with a quartz cuvette of 1 cm path length and a thermally controlled cell holder 32. The fluorescence emission (λem) spectra were recorded from 300 nm to 450 nm, with the path length for fluorescence excitation and emission of 5 mm, at an excitation wavelength (λex) of 280 nm.…”

Section: Methodsmentioning

confidence: 99%

Antigenic Characteristics of Rhinovirus Chimeras Designed in silico for En5hanced Presentation of HIV-1 gp41 Epitopes

Lapelosa

Arnold

Gallicchio

et al. 2010

Journal of Molecular Biology

View full text Add to dashboard Cite

The development of an effective AIDS vaccine remains the most promising long-term strategy to combat human immunodeficiency virus (HIV)/AIDS. Here, we report favorable antigenic characteristics of vaccine candidates isolated from a combinatorial library of human rhinoviruses displaying the ELDKWA epitope of the gp41 glycoprotein of HIV-1. The design principles of this library emerged from the application of molecular modeling calculations in conjunction with our knowledge of previously obtained ELDKWA-displaying chimeras, including knowledge of a chimera with one of the best 2F5-binding characteristics obtained to date. The molecular modeling calculations identified the energetic and structural factors affecting the ability of the epitope to assume conformations capable of fitting into the complementarity determining region of the ELDKWAbinding, broadly neutralizing human mAb 2F5. Individual viruses were isolated from the library following competitive immunoselection and were tested using ELISA and fluorescence quenching experiments. Dissociation constants obtained using both techniques revealed that some of the newly isolated chimeras bind 2F5 with greater affinity than previously identified chimeric rhinoviruses. Molecular dynamics simulations of two of these same chimeras confirmed that their HIV inserts were partially preorganized for binding, which is largely responsible for their corresponding gains in binding affinity. The study illustrates the utility of combining structure-based experiments with computational modeling approaches for improving the odds of selecting vaccine component designs with preferred antigenic characteristics. The results obtained also confirm the flexibility of HRV as a presentation vehicle for HIV epitopes and the potential of this platform for the development of vaccine components against AIDS.Keywords vaccine design; monoclonal antibody 2F5; chimeric virus; reorganization free energy; protein binding

show abstract

Section: Methodsmentioning

confidence: 99%

Antigenic Characteristics of Rhinovirus Chimeras Designed in silico for En5hanced Presentation of HIV-1 gp41 Epitopes

Lapelosa

Arnold

Gallicchio

et al. 2010

Journal of Molecular Biology

View full text Add to dashboard Cite

show abstract

“…Using replica exchange MD (REMD) simulations, Sanbonmatsu et al [6] estimated relatively low (0.8 kcal/mol) energy barriers for flipping of adenines in a ribosomal A-site model, consistent with fast flipping (that work predicted experimental values in the 0.5–5 kcal/mol range). Similarly, fluorescence anisotropy measurements performed on oligonucleotide A-site models suggest that the adenines stackdestack on the nanosecond timescale, with rotational correlation times of 0.540.15 ns for A1492 [72] and 0.470.09 ns for A1493 [73]. However, NMR carbon spin relaxation and relaxation dispersion studies [74] suggested micro- to millisecond timescales for full-range flips of the A-site nucleotides, with the nanosecond timescale corresponding to just partial destacking of the adenines.…”

Section: Resultsmentioning

confidence: 92%

Interplay of the Bacterial Ribosomal A-Site, S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

2014

View full text Add to dashboard Cite

The conformational properties of the aminoacyl-tRNA binding site (A-site), and its surroundings in the Escherichia coli 30S ribosomal subunit, are of great relevance in designing antibacterial agents. The 30S subunit A-site is near ribosomal protein S12, which neighbors helices h27 and H69; this latter helix, of the 50S subunit, is a functionally important component of an intersubunit bridge. Experimental work has shown that specific point mutations in S12 (K42A, R53A) yield hyper-accurate ribosomes, which in turn confers resistance to the antibiotic ‘paromomycin’ (even when this aminoglycoside is bound to the A-site). Suspecting that these effects can be elucidated in terms of the local atomic interactions and detailed dynamics of this region of the bacterial ribosome, we have used molecular dynamics simulations to explore the motion of a fragment of the E. coli ribosome, including the A-site. We found that the ribosomal regions surrounding the A-site modify the conformational space of the flexible A-site adenines 1492/93. Specifically, we found that A-site mobility is affected by stacking interactions between adenines A1493 and A1913, and by contacts between A1492 and a flexible side-chain (K43) from the S12 protein. In addition, our simulations reveal possible indirect pathways by which the R53A and K42A mutations in S12 are coupled to the dynamical properties of the A-site. Our work extends what is known about the atomistic dynamics of the A-site, and suggests possible links between the biological effects of hyper-accurate mutations in the S12 protein and conformational properties of the ribosome; the implications for S12 dynamics help elucidate how the miscoding effects of paromomycin may be evaded in antibiotic-resistant mutants of the bacterial ribosome.

show abstract

“…Crystallographic studies completed on ribosomal subunits or entire ribosomes in the presence of aminoglycosides provided near atomic resolution information on codon–anticodon interactions and the complex’s interaction with aminoglycosides (16–18). These studies served as a springboard for the Pilch and Hermann groups to develop fluorescence-based methods to study the effect that aminoglycosides have on A-site dynamics (19–22). Subsequent crystallographic studies by the Cate group on whole intact Escherichia coli ribosomes revealed that, in addition to the A-site, aminoglycosides bind helix 69 in 70S ribosomes, helping to explain how aminoglycosides inhibit ribosomal recycling (8).…”

Section: Introductionmentioning

confidence: 99%

Studying Modification of Aminoglycoside Antibiotics by Resistance-Causing Enzymes via Microarray

Disney

2011

Methods in Molecular Biology

View full text Add to dashboard Cite

Widespread bacterial resistance to antibiotics is a significant public health concern. To remain a step ahead of evolving bacteria, new methods to study resistance to antibacterials and to uncover novel antibiotics that evade resistance are urgently needed. Herein, microarray-based methods that have been developed to study aminoglycoside modification by resistance-causing enzymes are reviewed. These arrays can also be used to study the binding of aminoglycoside antibiotics to a mimic of their therapeutic target, the rRNA aminoacyl site (A-site), and how modification by resistance-causing enzymes affects their abilities to bind RNA.

show abstract

Use of 2-aminopurine as a fluorescent tool for characterizing antibiotic recognition of the bacterial rRNA A-site

Cited by 35 publications

References 30 publications

Antigenic Characteristics of Rhinovirus Chimeras Designed in silico for En5hanced Presentation of HIV-1 gp41 Epitopes

Antigenic Characteristics of Rhinovirus Chimeras Designed in silico for En5hanced Presentation of HIV-1 gp41 Epitopes

Interplay of the Bacterial Ribosomal A-Site, S12 Protein Mutations and Paromomycin Binding: A Molecular Dynamics Study

Studying Modification of Aminoglycoside Antibiotics by Resistance-Causing Enzymes via Microarray

Contact Info

Product

Resources

About