Modeling Amino Acid Side Chains in Proteins: ¹⁵N NMR Spectra of Guanidino Groups in Nonpolar Environments

Abstract

Natural-abundance 15N NMR spectroscopy on dodecylguanidine reveals solvent and protonation effects that model those that could occur for the arginine side chain in proteins. Our results demonstrate that the 15N chemical shifts of the terminal guanine nitrogens strongly depend on the solvent chosen for measurements. A polar H-bond-donating solvent like water has strongly deshielding effects on the neutral guanidine group (with the latter acting predominantly as an H-bond acceptor). As a result, a substantial upfield shift occurs when neutral guanidine is dissolved instead in a non-H-bonding solvent (chloroform). These solvent effects can be as large as those induced by protonation changes. This limits the ability of 15N chemical shifts to distinguish the protonation state of the arginine side chain, at least without specific knowledge of its environment. These results help to reconcile previous interpretations about the protonation state arg-82 in the M state of bacteriorhodopsin based on FTIR and 15N NMR spectroscopy. That is, contrary to earlier conclusions from solid-state NMR, the side chain of arg-82 could undergo a deprotonation between the bR and M states, but only if it also experienced a significant decrease in the H-bonding character and polarity of its environment. In fact, the average 15N chemical shift of the two Neta of arg-82 in bacteriorhodopsin's M intermediate (from the previous NMR measurements) is 17 ppm upfield from the corresponding value for the deprotonated arginine side chain in aqueous solution at pH >14, but only 3 ppm upfield from the value for deprotonated dodecylguanidine in chloroform.

Infrared and Visible Absolute and Difference Spectra of Bacteriorhodopsin Photocycle Intermediates

“…This is in stark contrast to the 15 N-NMR chemical shifts, which for deprotonated guanidines fall along an overlapping continuum with signals from protonated, but strongly H-bonded, guanidinium ions. 29 In IR spectra of model guanidine compounds, even very strong H-bonding of the protonated form always upshifts the pair of strong C-N stretch vibrations between 1600 and 1700 cm −1 , and never gives rise to a strong downshifted band near ~1550 cm −1 , where deprotonated guanidines have a strong characteristic vibrational frequency corresponding to line #5 of Table 3.…”

“…29 We can only make assignments of peaks #4 and #5 to Arg82 with confidence in the M intermediate, and even in this intermediate, only 30–50% of the intensity change at these positions is attributable to Arg82; the rest is more likely due to concomitant changes in amide I and amide II vibrations. Thus, when we label peaks at positions #4 and #5 in spectra of other intermediates besides BR and M, their assignment to Arg82 is only one of several possibilities.…”

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy

“…The two 15 N η chemical shifts of R52 obtained in this study are close to that for the guanidinium form, indicating that R52 is predominantly protonated. However, the partial burial of R52 in the low dielectric environment of the protein may affect the chemical shifts, potentially curbing the assignment of a unique protonation state.…”

Experimental and Computational Modeling of H-Bonded Arginine–Tyrosine Groupings in Aprotic Environments

“…The strongest characteristic IR bands of the protonated forms of both guanidinium (∼1670 cm –1 shoulder) and phenol (1514 cm –1 ) are present in DMSO (Figure 6A). The additional bands at 1642 and (especially) 1548 cm –1 are characteristic of deprotonated guanidine in either DMSO 12,17 or chloroform. 19 Meanwhile, characterisitic phenolate bands are observed at 1599 and 1494 cm –1 (Figure 6A), that is, shifted somewhat relative to this compound in MeOH (Figure 6B, 1602 and 1501 cm –1 ).…”