Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

Abstract

The ability to quantify the local electrostatic environment of proteins and protein/peptide assemblies is key to yielding a microscopic understanding of many biological interactions and processes. Herein, we show that the ester carbonyl stretching vibration of two non-natural amino acids, L-aspartic acid 4-methyl ester and L-glutamic acid 5-methyl ester, is a convenient and sensitive probe in this regard since its frequency correlates linearly with the local electrostatic field for both hydrogen-bonding and non-hydrogen-bonding environments. We expect that the resultant frequency-electric field map will find use in various applications. In addition, we show that, when situated in a non-hydrogen bonding environment, this probe can also be used to measure the local dielectric constant (ε). For example, applying it to amyloid fibrils formed by Aβ16-22 reveals that the interior of such β-sheet assemblies has a ε of ~5.6.

Simple method to introduce an ester infrared probe into proteins

“…This is because the YGGC*GG peptide, but not the YGGCGG peptide, gives rise to an IR band within the region of 1700-1750 cm 21 . 17 As expected, this ester carbonyl stretching vibrational band is broad and peaked at about 1720 cm 21 , which is similar to that observed for a short peptide containing a D M residue, due to hydrogen-bonding interactions with the solvent molecules. 17 In the second test of the cysteine alkylation strategy, we sought to incorporate the ME IR probe into two peptides that have been shown to form amyloid aggregates.…”

“…The study by Ivanova et al 27 indicated that the LVEALYL segment within the IB peptide can forms fibrils and may play an important role in the fibril formation of the full length insulin B chain. The second peptide correspond to a double mutant of Ab [16][17][18][19][20][21][22] (sequence: KCVK*FAE, hereafter referred to as the Ab peptide), with a cysteine at the 17 position and a lys-Nvoc (Fmoc-Lys(4,5-dimethoxy-2-nitro-benzyloxycarbonyl)-OH, K*) at the 19 position. Following the study of Measey and Gai, 28 the lys-Nvoc was used in this case to facilitate amyloid fibril formation.…”

“…1,5 For instance, to improve the structural and spatial resolution of IR measurements, non-natural amino acids consisting of a unique vibrational probe, such as a ACN, ASCN, or AN 3 moiety, have been utilized in various studies, including those concerning protein folding, 6 amyloid formation, 7 the structure and function of membrane proteins, 8,9 electrostatic field of enzymes, [10][11][12][13][14] and drug binding. 15,16 Recently, Pazos et al 17 have shown that the ester carbonyl stretching vibration is a useful and convenient site-specific IR probe of protein electrostatics because (1) its frequency is linearly correlated with the local electrostatic field, even when the carbonyl group is engaged in hydrogen-bonding interactions; (2) its frequency is located in the spectral range of 1700-1800 cm 21 , wherein no intrinsic protein vibrations show up at neutral pH; and (3) it has a relatively high extension coefficient. In particular, Pazos et al 17 have tested and demonstrated the utility of two ester-containing non-natural amino acids, L-aspartic acid 4-methyl ester (hereafter referred to as D M ) and L-glutamic acid 5-methyl ester (hereafter referred to as E M ), by incorporating them into various model peptide systems via solidphase peptide synthesis.…”

“…15,16 Recently, Pazos et al 17 have shown that the ester carbonyl stretching vibration is a useful and convenient site-specific IR probe of protein electrostatics because (1) its frequency is linearly correlated with the local electrostatic field, even when the carbonyl group is engaged in hydrogen-bonding interactions; (2) its frequency is located in the spectral range of 1700-1800 cm 21 , wherein no intrinsic protein vibrations show up at neutral pH; and (3) it has a relatively high extension coefficient. In particular, Pazos et al 17 have tested and demonstrated the utility of two ester-containing non-natural amino acids, L-aspartic acid 4-methyl ester (hereafter referred to as D M ) and L-glutamic acid 5-methyl ester (hereafter referred to as E M ), by incorporating them into various model peptide systems via solidphase peptide synthesis. In contrast, other non-natural amino acid-based IR probes, such as p-cyanophenylalanine 18,19 and p-azido-phenylalanine, 20,21 have been incorporated into large proteins in vivo via amber codon suppression.…”

Kinetics of peptide folding in lipid membranes

“…In addition, by using a photoresponsive structural constraint, it is possible to initiate a folding or unfolding process from a well‐selected conformational state. To improve the structural resolution of folding kinetic studies, we believe that the following approaches, many of which have already been used to study the equilibrium structures of membrane peptides and proteins, are promising: using (1) a local vibrational mode, such as that arising from an isotopically‐labeled amide unit or an unnatural amino acid side chain, , and resonance Raman scattering of a Trp probe, to gain site‐specific structural and environmental information; (2) the coupling between two site‐specific vibrators to probe tertiary interactions; (3) multiple fluorophores to simultaneously probe conformational changes of the protein of interest in different regions; (4) multi‐fluorophore FRET to reveal correlated conformational motions; and (5) various nonlinear techniques, such as two‐dimensional infrared, second harmonic generation, and sum frequency generation spectroscopy.…”