Evaluation of the Interaction between Bax and Hsp70 in Cells by Using a FRET System Consisting of a Fluorescent Amino Acid and YFP as a FRET Pair.

To gain insight into factors that lead to dissociation of Bax from a complex with Hsp70 during apoptosis, we recently constructed a fluorescence resonance energy transfer (FRET) system composed of the Hsp70-YFP (YFP=yellow fluorescent protein) fusion protein and fluorescent amino acid (ANAP=6-acetyl(naphthalen-2-ylamino)-2-aminopropanoic acid)-containing Bax (Bax-ANAP), which was produced by using the genetic code expansion technique. In the current study, the FRET system was employed to elucidate how brefeldin A (an endoplasmic reticulum stress inducer), chlorpromazine and apoptozole (lysosomal membrane destabilizers), bafilomycin A1 (an inhibitor of lysosomal acidification) as well as raptinal and Az-TPP-O3 (mitochondria-targeted apoptosis inducers) affect the interaction between Bax and Hsp70. Analyses of single live cell images together with results of co-immunoprecipitation assays reveal that brefeldin A, chlorpromazine, and apoptozole promote dissociation of the Bax/Hsp70 complex through activation of the activator BH3-only protein. However, the results show that bafilomycin A1, raptinal, and Az-TPP-O3 have no influence on the interaction of Bax with Hsp70. The combined observations made in the current and previous studies demonstrate that the FRET system consisting of Bax-ANAP and Hsp70-YFP is highly useful to understand apoptotic processes associated with the Bax-Hsp70 interaction.

Analysis of Protein-Protein Interaction in a Single Live Cell by Using a FRET System Based on Genetic Code Expansion Technology.

Hsp70 is known to directly bind to Bax for suppression of apoptosis. However, mechanisms on how Bax is dissociated from its complex with Hsp70 during apoptosis remain largely unknown. In the current study, we developed the efficient fluorescence resonance energy transfer (FRET) system which consisted of Hsp70-YFP and fluorescent amino acid (ANAP)-incorporated Bax, which was generated by using genetic code expansion technology, and applied the FRET system to elucidate mechanisms on how apoptosis-inducing substances dissociate Bax from Hsp70. Time-dependent analysis of single live cell images showed that Bax activators binding to Bax trigger sites inhibited the Bax-Hsp70 interaction but a Bax activator, which blocks phosphorylation of S184 via binding to the C-terminal S184 site, did not affect this interaction. Additionally, an inhibitor for Hsp70-Hsp40 interaction blocked the Bax-Hsp70 interaction. Furthermore, p53 activators promoted the dissociation of Bax from Hsp70 by reactivating p53 which disrupted the Bax-Hsp70 interaction. We also found that death ligands and a Bcl-2 inhibitor enhanced the dissociation of Bax from Hsp70 by activating activator BH3-only proteins. Results from this effort suggest that FRET systems consisting of the ANAP-incorporated protein and the YFP fusion protein will be valuable tools to gain an understanding of other types of protein-protein interactions.

Engineering a periplasmic binding protein for amino acid sensors with improved binding properties.

Periplasmic binding proteins (PBPs) are members of a widely distributed protein superfamily found in bacteria and archaea, and are involved in the cellular uptake of solutes. In this report, a leucine-binding PBP was engineered to detect l-Leu based on a fluorescence resonance energy transfer (FRET) change upon ligand binding. A fluorescent unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (CouA), was genetically incorporated into the protein as a FRET donor, and a yellow fluorescent protein (YFP) was fused with its N-terminus as a FRET acceptor. When CouA was incorporated into position 178, the sensor protein showed a 2.5-fold increase in the FRET ratio. Protein engineering significantly improved its substrate specificity, showing minimal changes in the FRET ratio with the other 19 natural amino acids and d-Leu. Further modification increased the sensitivity of the sensor protein (14-fold) towards l-Leu, and it recognized l-Met as well with moderate binding affinity. Selected mutant sensors were used to measure concentrations of l-Leu in a biological sample (fetal bovine serum) and to determine the optical purity of Leu and Met. This FRET-based sensor design strategy allowed us to easily manipulate the natural receptor to improve its binding affinity and specificity and to recognize other natural molecules, which are not recognized by the wild-type receptor. The design strategy can be applied to other natural receptors, enabling engineering receptors that sense biochemically interesting molecules.

Genetic incorporation of recycled unnatural amino acids.

The genetic incorporation of unnatural amino acids (UAAs) into proteins has been a useful tool for protein engineering. However, most UAAs are expensive, and the method requires a high concentration of UAAs, which has been a drawback of the technology, especially for large-scale applications. To address this problem, a method to recycle cultured UAAs was developed. The method is based on recycling a culture medium containing the UAA, in which some of essential nutrients were resupplemented after each culture cycle, and induction of protein expression was controlled with glucose. Under optimal conditions, five UAAs were recycled for up to seven rounds of expression without a decrease in expression level, cell density, or incorporation fidelity. This method can generally be applied to other UAAs; therefore, it is useful for reducing the cost of UAAs for genetic incorporation and helpful for expanding the use of the technology to industrial applications.

Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance.

FRET-based analysis of protein-nucleic acid interactions by genetically incorporating a fluorescent amino acid.

Protein-nucleic acid interaction is an important process in many biological phenomena. In this study, a fluorescence resonance energy transfer (FRET)-based protein-DNA binding assay has been developed, in which a fluorescent amino acid is genetically incorporated into a DNA-binding protein. A coumarin-containing amino acid was incorporated into a DNA-binding protein, and the mutant protein specifically produced a FRET signal upon binding to its cognate DNA labeled with a fluorophore. The protein-DNA binding affinity was then measured under equilibrium conditions. This method is advantageous for studying protein-nucleic acid interactions, because it is performed under equilibrium conditions, technically easy, and applicable to any nucleic acid-binding protein.

A fluorescence-based glycosyltransferase assay for high-throughput screening.

Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.

Development of specific l-methionine sensors by FRET-based protein engineering.

Amino acids are essential nutrients that are not only used as protein building blocks but are also involved in various biochemical processes and in the development of human diseases. Quantitative analysis of amino acids in complex biological samples is an important analytical process used for understanding amino acid biochemistry and diagnosis of human diseases. In this study, a protein sensor based on fluorescence resonance energy transfer (FRET) was designed for the quantitative analysis of l-Met, in which a fluorescent unnatural amino acid (CouA) and YFP were used as a FRET pair. A natural Met-binding protein (MetQ) was chosen as a sensor protein, and CouA and YFP were incorporated into the protein by genetic code expansion technology and genetic fusion. Among the four sites screened for CouA incorporation into MetQ, R189 was selected as the best site for l-Met sensing. The sensor protein (YFP-MetQ-R189CouA) showed a large FRET signal change (2.7-fold increase) upon l-Met binding. To improve amino acid specificity of the sensor protein, the ligand-binding site was engineered, and the mutant sensor (YFP-MetQ-R189CouA-H88F) with the H88F mutation was identified, which showed no FRET signal change with d-Met and l-Gln at 50 µM concentration and retained the maximum FRET signal change with l-Met. The optimized sensor protein was evaluated for biochemical applications. l-Met concentration in FBS and optical purity in a mixture of d- and l-Met were successfully determined. Because l-Met is biochemically important owing to its involvement in cancer cell growth and autophagy, the sensor protein would be useful for quantitative analysis of l-Met in a complex biological sample. In addition, the design strategy used in this study can be applied to other small molecule-binding proteins for the development of protein sensors for important biomolecules.

Construction of Bacterial Cells with an Active Transport System for Unnatural Amino Acids.

Engineered organisms with an expanded genetic code have attracted much attention in chemical and synthetic biology research. In this work, engineered bacterial organisms with enhanced unnatural amino acid (UAA) uptake abilities were developed by screening periplasmic binding protein (PBP) mutants for recognition of UAAs. A FRET-based assay was used to identify a mutant PBP (LBP-AEL) with excellent binding affinity ( Kd ≈ 500 nM) to multiple UAAs from 37 mutants. Bacterial cells expressing LBP-AEL showed up to 5-fold enhanced uptake of UAAs, which was determined by genetic incorporation of UAAs into a green fluorescent protein and measuring UAA concentration in cell lysates. To the best of our knowledge, this work is the first report of engineering cellular uptake of UAAs and could provide an impetus for designing advanced unnatural organisms with an expanded genetic code, which function with the efficiency comparable to that of natural organisms. The system would be useful to increase mutant protein yield from lower concentrations of UAAs for industrial and large-scale applications. In addition, the techniques used in this report such as the sensor design and the measurement of UAA concentration in cell lysates could be useful for other biochemical applications.

Truncated TALE-FP as DNA Staining Dye in a High-salt Buffer.

Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators.

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition.

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Fast, exact, and non-destructive diagnoses of contact failures in nano-scale semiconductor device using conductive AFM.

We fabricated a novel in-line conductive atomic force microscopy (C-AFM), which can analyze the resistive failures and examine process variance with an exact-positioning capability across the whole wafer scale in in-line DRAM fabrication process. Using this in-line C-AFM, we introduced a new, non-destructive diagnosis for resistive failure in mobile DRAM structures. Specially, we focused on the self-aligned contact (SAC) process, because the failure of the SAC process is one of the dominant factors that induces the degradation of yield performance, and is a physically invisible defect. We successfully suggested the accurate pass mark for resistive-failure screening in the fabrication of SAC structures and established that the cause of SAC failures is the bottom silicon oxide layer. Through the accurate pass mark for the SAC process configured by the in-line C-AFM analyses, we secured a good potential method for preventing the yield loss caused by failures in DRAM fabrication.

Measurement system of the refractive power of spherical and sphero-cylindrical lenses with the magnification ellipse fitting method.

This paper proposes a new measurement system for measuring the refractive power of spherical and sphero-cylindrical lenses with a six-point light source, which is composed of a light emitting diode and a six-hole pattern aperture, and magnification ellipse fitting method. The position of the six light sources is changed into a circular or elliptical form subjected to the lens refractive power and meridian rotation angle. The magnification ellipse fitting method calculates the lens refractive power based on the ellipse equation with magnifications that are the ratios between initial diagonal lengths and measured diagonal lengths of the conjugated light sources changed by the target lens. The refractive powers of the spherical and sphero-cylindrical lenses certified in the Korea Research Institute of Standard and Science were measured to verify the measurement performance. The proposed method is estimated to have a repeatability of +/-0.01 D and an error value below 1%.