True Origin of Amide I Shifts Observed in Protein Spectra Obtained with Sum Frequency Generation Spectroscopy

Abstract

Accurate determination of protein structure at interfaces is critical for understanding protein interactions, which is directly relevant to a molecular-level understanding of interfacial proteins in biology and medicine. Vibrational sum frequency generation (VSFG) spectroscopy is often used for probing the protein amide I mode, which reports protein structures at interfaces. Observed peak shifts are attributed to conformational changes and often form the foundation of hypotheses explaining protein working mechanisms. Here, we investigate structurally diverse proteins using conventional and heterodyne-detected VSFG (HD-VSFG) spectroscopy as a function of solution pH. We reveal that blue-shifts of the amide I peak observed in conventional VSFG spectra upon lowering the pH are governed by the drastic change of the nonresonant contribution. Our results highlight that connecting changes in conventional VSFG spectra to conformational changes of interfacial proteins can be arbitrary, and that HD-VSFG measurements are required to draw unambiguous conclusions about structural changes in biomolecules.

Figure 1

Conventional VSFG spectra of 1g/L TmAFP (a), HStarB (b), and BSA (c) at the deuterated water/air interface for different bulk pDs. Acidic, near IEP, and basic indicate pD ∼3, ∼6, and ∼11, respectively. The ionic strength was held at 300 mM by adding NaCl to the solution containing proteins. Schematic pictures of the bulk protein structures are displayed on the top of the VSFG spectra. Note that the VSFG intensity spectra presented here were obtained directly from the conventional VSFG method.

Figure 2

Imaginary and real parts of HD-VSFG spectra (Imχ⁽²⁾ and Reχ⁽²⁾, respectively) of 1g/L TmAFP, HStarB, and BSA at the deuterated water/air interface for different bulk pDs. Acidic, near IEP, and basic indicate pD ∼3, ∼6, and ∼11, respectively. The bottom panel shows the constructed |χ⁽²⁾|² to compare with the conventional VSFG spectra shown in Figure 1. Note that the presented constructed |χ⁽²⁾|² is calculated via |χ⁽²⁾|² = |Reχ⁽²⁾|² + |Imχ⁽²⁾ |². All HD-VSFG spectra were measured in deuterated water with an ionic strength of 300 mM NaCl.

Figure 3

(a) Real part of HD-VSFG spectra (Reχ⁽²⁾) at the water–air, HCl solution–air, and NaOH solution–air interfaces. (b) Reχ⁽²⁾ spectra at the water–lipid interfaces. The composition of the lipid is xDOPC+(1 – x)DPPG/DPTAP. The surface charge is computed from the charge of the lipid. (c) Amplitude of the nonresonant part versus surface charge. The nonresonant amplitude was obtained by averaging the amplitude in panel (b) from 2000 to 2200 cm^–1. Error bar shows standard deviation. The line is a sigmoidal fit to guide the eye. The arrows with color gradient in (b) and (c) indicate the charge densities.

Figure 4

Effects of nonresonant contributions χ_NR⁽²⁾ on the constructed |χ⁽²⁾|² spectra for TmAFP, HstarB, and BSA. The offsets Δχ_NR⁽²⁾ of the nonresonant term were estimated to be ∼ ± 0.018 from Figure 3. The solid and dashed black lines in the upper panel are the original real and imaginary parts of near IEP χ⁽²⁾ spectra from Figure 2. The blue and red lines represent the positive and negative enhancements of the nonresonance signal. The dashed lines in the lower panels indicate the middle points of the peak fwhm in the |χ⁽²⁾|² spectra.