Ultrafast Laser-Probing Spectrocopy for Studying Molecular Structure of Polymeric Proteins

AbstractWe report the development of a new technique to screen protein crystallinity quantitatively based on laser-probing spectroscopy with sub-picosecond resolution. First, we show theoretically that the temperature dependence of the refractive index of a polymeric protein is correlated to its crystallinity. Then, we performed time-domain thermo-transmission experiments on purified semi-crystalline proteins, both native and recombinant (i.e., silk and squid ring teeth), and also on intactE. colicells bearing overexpressed recombinant protein. Our results demonstrate, for the first time, quantification of crystallinity in real time for polymeric proteins. Our approach can potentially be used for screening an ultra-large number of polymeric proteinsin vivo.

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Ultrafast laser-probing spectroscopy for studying molecular structure of protein aggregates

The Analyst ◽

10.1039/c6an02570f ◽

2017 ◽

Vol 142 (9) ◽

pp. 1434-1441 ◽

Cited By ~ 5

Author(s):

Huihun Jung ◽

Chester J. Szwejkowski ◽

Abdon Pena-Francesch ◽

John A. Tomko ◽

Benjamin Allen ◽

...

Keyword(s):

Molecular Structure ◽

Protein Aggregation ◽

Protein Aggregates ◽

Ultrafast Laser ◽

New Technique ◽

Laser Probing ◽

A New Technique

We report the development of a new technique to screen protein aggregation based on laser-probing spectroscopy with sub-picosecond resolution.

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AN ANALYSIS OF THE THZ FREQUENCY SIGNATURES IN THE CELLULAR COMPONENTS OF BIOLOGICAL AGENTS

International Journal of High Speed Electronics and Systems ◽

10.1142/s012915640700445x ◽

2007 ◽

Vol 17 (02) ◽

pp. 225-237 ◽

Cited By ~ 8

Author(s):

ALEXEI BYKHOVSKI ◽

TATIANA GLOBUS ◽

TATYANA KHROMOVA ◽

BORIS GELMONT ◽

DWIGHT WOOLARD

Keyword(s):

Molecular Structure ◽

Structural Similarity ◽

Md Simulations ◽

Detection Capability ◽

Dna Structures ◽

E Coli ◽

Cellular Components ◽

First Time ◽

Insight Into ◽

Specific Contribution

The development of an effective biological (bio) agent detection capability based upon terahertz (THz) frequency absorption spectra will require insight into how the constituent cellular components contribute to the overall THz signature. In this work, the specific contribution of ribonucleic acid (RNA) to THz spectra is analyzed in detail. Previously, it has only been possible to simulate partial fragments of the RNA (or DNA) structures due to the excessive computational demands. For the first time, the molecular structure of the entire transfer RNA (tRNA) molecule of E. coli was simulated and the associated THz signature was derived theoretically. The tRNA that binds amino acid tyrosine (tRNAtyr) was studied. Here, the molecular structure was optimized using the potential energy minimization and molecular dynamical (MD) simulations. Solvation effects (water molecules) were also included explicitly in the MD simulations. To verify that realistic molecular signatures were simulated, a parallel experimental study of tRNAs of E. coli was also conducted. Two very similar molecules, valine and tyrosine tRNA were investigated experimentally. Samples were prepared in the form of water solutions with the concentrations in the range 0.01-1 mg/ml. A strong correlation of the measured THz signatures associated with valine tRNA and tyrosine tRNA was observed. These findings are consistent with the structural similarity of the two tRNAs. The calculated THz signature of the tyrosine tRNA of E. coli reproduces many features of our measured spectra, and, therefore, provides valuable new insights into bio-agent detection.

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Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins

Journal of Bacteriology ◽

10.1128/jb.184.3.629-635.2002 ◽

2002 ◽

Vol 184 (3) ◽

pp. 629-635 ◽

Cited By ~ 79

Author(s):

J. M. Nieto ◽

C. Madrid ◽

E. Miquelay ◽

J. L. Parra ◽

S. Rodríguez ◽

...

Keyword(s):

Recombinant Protein ◽

Protein A ◽

Nitrilotriacetic Acid ◽

Mass Spectrometry Analysis ◽

Spectrometry Analysis ◽

E Coli ◽

Protein Protein Interaction ◽

New Family ◽

Missense Mutant ◽

First Time

ABSTRACT Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin α-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni2+-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha′ proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

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DNA Gap Repair-Mediated Site-Directed Mutagenesis is Different from Mandecki and Recombineering Approaches

10.1101/313155 ◽

2018 ◽

Author(s):

George T. Lyozin ◽

Luca Brunelli

Keyword(s):

Dna Sequence ◽

Dna Sequences ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Products ◽

E Coli ◽

Health And Disease ◽

First Time ◽

Disease Understanding

AbstractSite-directed mutagenesis allows the generation of mutant DNA sequences for downstream functional analysis of genetic variants involved in human health and disease. Understanding the mechanisms of different mutagenesis methods can help select the best approach for specific needs. We compared three different approaches for in vivo site-directed DNA mutagenesis that utilize a mutant single-stranded DNA oligonucleotide (ssODN) to target a wild type DNA sequence in the host Escherichia coli (E. coli). The first method, Mandecki, uses restriction nucleases to introduce a double stranded break (DSB) into a DNA sequence which needs to be denatured prior to co-transformation. The second method, recombineering (recombination-mediated genetic engineering), requires lambda red gene products and a mutant ssODN with homology arms of at least 20 nucleotides. In a third method described here for the first time, DNA gap repair, a mutant ssODN targets a DNA sequence containing a gap introduced by PCR. Unlike recombineering, both DNA gap repair and Mandecki can utilize homology arms as short as 10 nucleotides. DNA gap repair requires neither red gene products as recombineering nor DNA denaturation or nucleases as Mandecki, and unlike other methods is background-free. We conclude that Mandecki, recombineering, and DNA gap repair have at least partly different mechanisms, and that DNA gap repair provides a new, straightforward approach for effective site-directed mutagenesis.

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Quantification of In Vitro and In Vivo Cryptosporidium parvum Infection by Using Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.00189-06 ◽

2006 ◽

Vol 72 (6) ◽

pp. 4484-4488 ◽

Cited By ~ 9

Author(s):

Nihal T. Godiwala ◽

Alain Vandewalle ◽

Honorine D. Ward ◽

Brett A. Leav

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Absolute Quantification ◽

Host Tissue ◽

Quantitative Real Time Pcr ◽

A New Technique ◽

First Time ◽

Laboratory Models

ABSTRACT Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.

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Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter-protein

10.1101/2020.07.23.216291 ◽

2020 ◽

Author(s):

Clara D. Wang ◽

Rachel Mansky ◽

Hannah LeBlanc ◽

Chandra M. Gravel ◽

Katherine E. Berry

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Regulate Gene Expression ◽

E Coli ◽

Binding Energetics ◽

Response To Stress ◽

First Time ◽

Regulate Gene

ABSTRACTNon-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system, and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system, and will facilitate exploration of the binding mechanisms of these interactions.

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Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of aPlasmodiumRegion I-Plus Peptide

BioMed Research International ◽

10.1155/2014/261631 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Xuemei Lu ◽

Xiaobao Jin ◽

Yanting Huang ◽

Jie Wang ◽

Juan Shen ◽

...

Keyword(s):

Recombinant Protein ◽

Current Treatment ◽

Biologically Active ◽

Liver Targeting ◽

E Coli ◽

Final Yield ◽

Pcr Method ◽

Overlapping Extension Pcr

Interferon alpha (IFNα) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFNαis the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFNαmay account for its lowin vivobiological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFNα2b with aPlasmodiumregion I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression inE. coliBL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentialityin vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.

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Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish

Biomolecules ◽

10.3390/biom10091202 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1202

Author(s):

Chung-Der Hsiao ◽

Hsin-Hui Wu ◽

Nemi Malhotra ◽

Yen-Ching Liu ◽

Ying-Hsuan Wu ◽

...

Keyword(s):

Copper Ions ◽

Copper Ion ◽

Biological Properties ◽

In Vivo Model ◽

Glutathione S Transferase ◽

Bacterial Fermentation ◽

E Coli ◽

Cu Complex ◽

First Time

In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO4-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time.

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In vivo amyloid-like fibrils produced under stress

10.1101/2021.02.02.429251 ◽

2021 ◽

Author(s):

Natália A. Fontana ◽

Ariane D. Rosse ◽

Anthony Watts ◽

Paulo S. R. Coelho ◽

Antonio J. Costa-Filho

Keyword(s):

Direct Detection ◽

Size Exclusion ◽

Fluorescence Lifetime Imaging ◽

E Coli ◽

Lifetime Imaging ◽

Exclusion Chromatography ◽

Higher Temperature ◽

Β Sheets ◽

First Time

AbstractThe participation of amyloids in neurodegenerative diseases and in functional processes has triggered the quest for methods allowing their direct detection in vivo. Despite the plethora of data, those methods are still lacking. We used the autofluorescence from the extended β-sheets of amyloids to follow fibrillation of S. cerevisiae Golgi Reassembly and Stacking Protein (Grh1). Grh1 has been implicated in starvation-triggered unconventional protein secretion (UPS) and here we suggest the idea of its participation also in heat shock response (HSR). Fluorescence Lifetime Imaging (FLIM) was used to detect fibril autofluorescence in cells (E. coli and yeast) under stress (starvation and higher temperature). The formation of Grh1 large complexes under stress was further supported by size exclusion chromatography and ultracentrifugation. Our data show the first-time in vivo detection of amyloids without the use of extrinsic probes as well as bring new perspectives on the participation of Grh1 in UPS and HSR.

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Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes

Eukaryotic Cell ◽

10.1128/ec.00147-14 ◽

2014 ◽

Vol 13 (9) ◽

pp. 1222-1231 ◽

Cited By ~ 21

Author(s):

Patrick C. Thiaville ◽

Basma El Yacoubi ◽

Ludovic Perrochia ◽

Arnaud Hecker ◽

Magali Prigent ◽

...

Keyword(s):

Mitochondrial Pathway ◽

Stem Loop ◽

Content Type ◽

E Coli ◽

Functional Conservation ◽

The Core ◽

First Time ◽

Keops Complex

ABSTRACT Threonylcarbamoyladenosine (t 6 A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t 6 A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t 6 A machinery of Saccharomyces cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332–6346, 2013, http://dx.doi.org/10.1093/nar/gkt322 ). Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP), while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t 6 A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The import of Sua5p into the mitochondria is required for this organelle to be functional, since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. This minimal t 6 A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli . The data revealed a potential for TC-AMP channeling in the t 6 A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t 6 A and bring additional advancement in our understanding of the reaction mechanism.

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