Orthogonal translation with 5‐cyanotryptophan as an infrared probe for local structural information, electrostatics, and hydrogen bonding

Abstract

Orthogonal translation is an efficient tool that provides many valuable spectral probes capable of covering different parts of the electromagnetic spectrum and thus enabling parameterization of various structural and dynamic phenomena in proteins. In this context, nitrile‐containing tryptophan analogs are very useful probes to study local electrostatics and hydrogen bonding in both rigid and dynamic environments. Here, we report a semi‐rational approach to engineer a tyrosyl‐tRNA synthetase (TyrRS) variant of Methanocaldococcus jannaschii capable of incorporating 5‐cyanotryptophan (5CNW) via orthogonal translation. We combined one round of the well‐established positive selection system with saturation mutagenesis at preselected TyrRS positions, resulting in a novel 5CNW‐specific enzyme that also exhibits high substrate tolerance to other aromatic noncanonical amino acids. We demonstrated the utility of our orthogonal pair by inserting 5CNW into the cyanobacteriochrome Slr1393g3, a bilin‐binding photosensor of the phytochrome superfamily. The nitrile (CN) group of the inserted 5CNW provides non‐invasive labeling in the local structural context while yielding information on local electrostatics and hydrogen bonding by IR spectroscopy. 5CNW is a versatile probe that can be used for both static and dynamic measurements.

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…Recently, Lin and co-workers successfully incorporated several Trp derivatives, such as 6CN-Trp, into Trp-rich proteins, using the orthogonal chPheRS system. Similarly, Budisa and co-workers incorporated 5CN-Trp into a photosensitive protein from phytochrome superfamily via orthogonal translation.…”

Triple-Bond Vibrations: Emerging Applications in Energy and Biological Sciences

“…Because of the proven sensitivity of ν CN to local electrostatic field and hydrogen-bonding (H-bonding) interactions, the CN stretching vibration has long been used to interrogate various biological questions and processes through the use of nitrile-containing nonnatural amino acids, ,,− ranging from protein hydration and conformational dynamics to protein electrostatics. , Several recent studies (since 2020) further elaborated the biological and biophysical utilities of the CN stretching vibration. For example, (1) Boxer and co-workers showed that its integrated oscillator strength (or intensity) can be used to quantify the electric field projected onto the CN bond, in both protic and aprotic environments; (2) Liu et al demonstrated the utility of this vibrational mode to probe solvation dynamics (Figure ); (3) Zhong and Ding and co-workers used it to delineate the aggregation kinetics and mechanism of an amyloidogenic fragment of protein SOD1, as well as to assess photoinduced electron transfer dynamics in a peptide construct; (4) Hildebrandt, Budisa, and co-workers used it to assess how the local electric field changes during the photoconversion process of the dark-to-photoactivated state of the bathy phytochrome Agp2; (5) Fuertes and co-workers used it to provide site-specific structural and dynamic information about the key kinetic steps associated with the dark reversion process of the bacterial transcription factor EL222; (6) Thielges and co-workers employed it and 2D IR spectroscopy to study how structural heterogeneity and dynamics of the archetypical enzyme cytochrome P450cam differentiate different substrate recognition; and (7) Hunt and co-workers used 2D IR spectroscopy and both the carbonyl (CO) and cyanide (CN) stretching bands of the Fe­(CO)­(CN) 2 unit at the active site of the group 1 [NiFe] hydrogenase Ec Hyd-1 to examine the role of the protein scaffold in controlling the active site environment. Furthermore, the CN stretching vibration has been used to investigate, for example, the effect of Hofmeister anions on the strength of water’s H-bonding interactions, the local acidity in condensed phase, and potential distribution across model membranes .…”