Influence of histidine tag attachment on picosecond protein dynamics

Abstract

Polyhistidine affinity tags are routinely employed as a convenient means of purifying recombinantly expressed proteins. A tacit assumption is commonly made that His tags have little influence on protein structure and function. Attachment of a His tag to the N-terminus of the robust globular protein myoglobin leads to only minor changes to the electrostatic environment of the heme pocket, as evinced by the nearly unchanged Fourier transform infrared spectrum of CO bound to the heme of His-tagged myoglobin. Experiments employing two-dimensional infrared vibrational echo spectroscopy of the heme-bound CO, however, find that significant changes occur to the short time scale (picoseconds) dynamics of myoglobin as a result of His tag incorporation. The His tag mainly reduces the dynamics on the 1.4 ps time scale and also alters protein motions of myoglobin on the slower, >100 ps time scale, as demonstrated by the His tag's influence on the fluctuations of the CO vibrational frequency, which reports on protein structural dynamics. The results suggest that affinity tags may have effects on protein function and indicate that investigators of affinity-tagged proteins should take this into consideration when investigating the dynamics and other properties of such proteins.

Figure 1

Structure of sperm whale myoglobin (pdb 1bzr) with bound CO ligand. Site of N-terminal His₆ affinity tag placement shown by red arrow.

Figure 2

FT-IR spectra of (A) MbCO and (B) His₆MbCO. Gaussian fits are shown as dashed lines. The inset in (B) shows a 30X expanded view (1960-1975 cm^-1) of the A₀ band.

Figure 3

2D IR spectra of MbCO (upper panels) and His₆MbCO (lower panels) for various T_w times. A total of 20 contour lines are shown.

Figure 4

CLS decay curves and corresponding exponential fits for MbCO (blue) and His₆MbCO (red).