An orthogonal tRNA/aminoacyl-tRNA synthetase pair has been evolved that makes it possible to selectively and efficiently incorporate para-cyanophenylalanine (pCNPhe) into proteins in E. coli at sites specified by the amber nonsense codon, TAG. Substitution of pCNPhe for histidine-64 in myoglobin (Mb) affords a sensitive vibrational probe of ligand binding. This methodology provides a useful infrared reporter of protein structure, biomolecular interactions, and conformational changes.

We have developed a single-plasmid system for the efficient bacterial expression of mutant proteins containing unnatural amino acids at specific sites designated by amber nonsense codons. In this system, multiple copies of a gene encoding an amber suppressor tRNA derived from a Methanocaldococcus jannaschii tyrosyl-tRNA (MjtRNATyrCUA) are expressed under control of the proK promoter and terminator, and a gene encoding the desired mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) is expressed under control of a mutant glnS (glnS') promoter.

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by co-expressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy) phenyl) propanoic acid (OCF 3 Phe), 13 C/ 15 N-labeled p-methoxyphenylalanine (OMePhe), and 15 N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3 Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of E. coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in 1 H-15 N, 1 H-13 C HSQC and 19 F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3 Phe mutants of an active site tyrosine inhibited binding; incorporating 15 N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.

Activating proteins with light: A photocaged tyrosine was genetically encoded in E. coli in response to the amber codon TAG. Substitution of Tyr 503 in the active site of β‐galactosidase allowed photoactivation of this enzyme in vitro or directly in bacteria with 360‐nm light. This method should allow photoregulation of the activity of a variety of biological processes including transcription, signal transduction, and cellular trafficking.

The measurement of velocity fields of a plunging wave impacting on a structure in a two-dimensional wave tank was investigated experimentally. As the wave impinged and overtopped the structure, a large highly aerated region was created in front of the structure and on top of the structure. The broken wave in front of the structure and associated greenwater on top of the structure are highly aerated containing not only a large number of bubbles but also very large sizes of bubbles. The highly aerated bubbly flow caused the traditional particle image velocimetry (PIV) technique to fail due to the uncontrollable scattering of laser light. A modified PIV method, called bubble image velocimetry (BIV), was introduced by directly using bubbles as the tracer and measuring the bubble velocity by correlating the ‘texture’ of the bubble images. No laser light sheet was needed while the depth of field was limited to minimize the error. Velocity measurements using BIV and fibre optic reflectometer were compared to validate the BIV technique. While the fluid velocity in the region where no or few bubbles exist can be successfully obtained using PIV, the velocity in the high void fraction region can be measured using BIV. Therefore, BIV can be seen as a complementary technique for PIV. The use of BIV is essential in the studied problem here due to the fact that in the vicinity of the structure the flow is almost entirely bubbly flow. From both the PIV and BIV measurements, it was found that the maximum fluid particle velocity as well as the bubble velocity in front of the structure during the impinging process is about 1.5 times the phase speed of the waves.