Simple method to introduce an ester infrared probe into proteins

Abstract

The ester carbonyl stretching vibration has recently been shown to be a sensitive and convenient infrared (IR) probe of protein electrostatics due to the linear dependence of its frequency on local electric field. While an ester moiety can be easily incorporated into peptides via solid-phase synthesis, currently there is no method available to site-specifically incorporate it into a large protein. Herein, we show that it is possible to use a cysteine alkylation reaction to achieve this goal and demonstrate the feasibility of this simple method by successfully incorporating a methyl ester group (CH COOCH ) into a model peptide (YGGCGG), two amyloid-forming peptides derived from the insulin B chain and Aβ, and bovine serum albumin (BSA). IR results obtained with those peptide and protein systems further confirm the utility of this vibrational probe in monitoring, for example, the structural integrity of amyloid fibrils and ligand binding-induced changes in protein local hydration status.

Vibrational Approach to the Dynamics and Structure of Protein Amyloids

“…The same probe was also introduced to another Aβ 16–22 peptide derivative to discriminate the hydration status of local residues for dry fibrils and fibrils in aqueous solution by measuring the ester carbonyl stretching vibration [176]. Similarly, a methyl ester group was also introduced to the side chain of the cysteine residue of amyloidogenic peptides via cysteine alkylation, to successfully probe the local hydration state and the structural integrity of the amyloid fibrils [177]. These studies highlight the potential utility of the ester carbonyl stretching vibration as a convenient means for structural determination of amyloids fibrils and local environmental information along the aggregation pathway.…”

Local Electric Fields in Aqueous Electrolytes

“… is the solvent reaction field, is the dipole moment of the solute, ε r is the relative static permittivity of the solvent (ε r = 78.54 for pure water at 25 °C), n is the refractive index of the solute, and a is the radius of the cavity occupied by the solute. The local reaction field couples back to the solute, which gives rise to a Stark shift in the optical and vibrational spectra of polar chromophores. − As such, modulating the solvent’s polarity can lead to pronounced changes in the color of visible dyes through an effect known as optical solvatochromism. − Moreover, vibrational solvatochromism has been observed in molecular probes with CO and CN bonds. − This Stark shift has been exploited to estimate electric fields in catalysis. − …”

Inconsistent hydrogen bond-mediated vibrational coupling of amide I

“…To overcome such difficulties, the structural and environmental properties of biomolecules have been investigated using “vibrational probes”. 21–26…”

“…To overcome such difficulties, the structural and environmental properties of biomolecules have been investigated using "vibrational probes". [21][22][23][24][25][26] Many biomolecules contain "amide I", "amide II", "amide III", and "amide A" modes of vibration. However, the amide-I mode is studied widely as a vibrational probe.…”

“…[33][34][35][36][37][38] In particular, vibrational spectroscopic measurements of the amide-I band are used to monitor shis in the transition frequency, which is sensitive to the local electric elds as well as interactions with specic "guest molecules". [24][25][26][27] Many studies have focused on the relationship between vibrational couplings and conformational dynamics of proteins/ peptides. [34][35][36][37][38][39][40][41] Various theoretical methodologies and multidimensional IR-spectroscopy methods have been employed to investigate the vibrational coupling and structural details of biological systems.…”