Two-dimensional IR spectroscopy of protein dynamics using two vibrational labels: a site-specific genetically encoded unnatural amino acid and an active site ligand

Abstract

Protein dynamics and interactions in myoglobin (Mb) were characterized via two vibrational dynamics labels (VDLs): a genetically incorporated site-specific azide (Az) bearing unnatural amino acid (AzPhe43) and an active site CO ligand. The Az-labeled protein was studied using ultrafast two-dimensional infrared (2D IR) vibrational echo spectroscopy. CO bound at the active site of the heme serves as a second VDL located nearby. Therefore, it was possible to use Fourier transform infrared (FT-IR) and 2D IR spectroscopic experiments on the Az in unligated Mb and in Mb bound to CO (MbAzCO) and on the CO in MbCO and MbAzCO to investigate the environment and motions of different states of one protein from the perspective of two spectrally resolved VDLs. A very broad bandwidth 2D IR spectrum, encompassing both the Az and CO spectral regions, found no evidence of direct coupling between the two VDLs. In MbAzCO, both VDLs reported similar time scale motions: very fast homogeneous dynamics, fast, ∼1 ps dynamics, and dynamics on a much slower time scale. Therefore, each VDL reports independently on the protein dynamics and interactions, and the measured dynamics are reflective of the protein motions rather than intrinsic to the chemical nature of the VDL. The AzPhe VDL also permitted study of oxidized Mb dynamics, which could not be accessed previously with 2D IR spectroscopy. The experiments demonstrate that the combined application of 2D IR spectroscopy and site-specific incorporation of VDLs can provide information on dynamics, structure, and interactions at virtually any site throughout any protein.

Figure 1

Structure of MbCO (PDB ID 1BZR) showing the local environment of CO and the Az VDL of AzPhe43.

Figure 2

FT-IR spectra of CO in (A) MbCO and (B) MbAzCO and of Az in (C) MbAz and (D) MbAzCO. Experimental data are shown in black. For A and B, the individual Gaussian fits are shown in blue, and their sum is shown in red. In C and D, the Voigt and Gaussian fits to the data are shown in red and blue, respectively. The residual spectra from subtraction of the Voigt fits of the main bands from the experimental spectra are shown in green.

Figure 3

2D IR spectra of CO in MbCO (top panels) and MbAzCO (bottom panels) at two T_w’s.

Figure 4

2D IR spectra of Az in MbAz (top panels) and MbAzCO (bottom panels) at two T_w’s.

Figure 5

2D IR spectra of MbAzCO at T_w of 300 fs encompassing both Az and CO spectral regions. The region around the CO peaks (lower left corner) has been divided by 25 so that the CO bands will have approximately the same amplitude as the Az bands. The red circles show the locations where off-diagonal peaks should be plainly visible if the CO and Az vibrations were directly coupled.

Figure 6

Upper panel: CLS decays (symbols) and exponential fits (lines) for CO in MbCO (red) and MbAzCO (blue). Lower panel: CLS decays (symbols) and exponential fits (lines) for Az in MbAz (red) and MbAzCO (blue).