Speci�ically, small molecule electric �ield-TDM calibrations remain robust when nitrile TDMs and MD electric �ields for both aprotic solvents and water are included. This TDMbased VSE enables extraction of FC≡N,TDM's sensed by the -C≡N via the peak area (analogous to FC≡N,FREQ's obtained via an electric �ield-frequency calibration), circumventing the interpretation issues for the nitrile frequency in H-bonding environments. What is more, a joint analysis of TDMs and frequency shifts enables quanti�ication of the H-bonding blueshift. 24 To demonstrate the applicability of using the TDM to extract FC≡N,TDM's in proteins, we incorporated the nitrilecontaining noncanonical amino acid o-cyanophenylalanine (oCNF) into photoactive yellow protein (PYP) via amber suppression at four native phenylalanine sites (positions 28, 62, 92, and 96) and obtained high-resolution crystal structures (Figure ). In the structures, F62oCNF and F96oCNF are in nonpolar environments, while F28oCNF and F92oCNF each possess at least one H-bond donor (de�ined by heavy-atom distances of < 4.0 AÅ for consistency i "FXXoCNF" (where XX = 28, 62, 92, or 96) is used as shorthand for "the nitrile of FXXoCNF." with de�initions in MD simulations (vide infra); Figure ). i Electric �ield-TDM calibrations were obtained for the model compound o-tolunitrile (oTN) with both FC and POL MD. Interpreting the protein -C≡N absorption intensities in IR spectra with the POL MD calibration revealed that the �ields are as large as -60 MV/cm (F92oCNF) and as small as -9 MV/cm (F96oCNF; Table ; Figure ). ii This range of 50 MV/cm is similar to the difference experienced for oTN in hexanes and water, highlighting the substantially different noncovalent interactions that occur within the protein. In this study, we utilize this set of PYP variants to revisit the question of how accurately local electric �ields are modelled within diverse protein environments by simulating the proteins with FC and POL MD FFs. We compare experimentally-derived FC≡N,TDM's with computationally-derived FC≡N,MD's and explore the structural and parametric rationales which underlie the observed (dis)agreements. Particular attention is paid to comparisons for F28oCNF and F92oCNF, the nitriles with crystallographic H-bond donors and substantial H-bonding blueshifts, whose electric �ields were not experimentally assessable prior to the new TDM-based VSE.