Probing the Stepwise Unfolding of Bovine Serum Albumin Using 2D Correlation Raman Spectroscopic Analysis

Abstract

Protein denaturation involves a change in the protein structure with the loss of activity, which proceeds via various intermediates. The possible intermediate structures account largely for understanding the process of unfolding. Hence, considerable attention is required to characterize partially unfolded protein states and to gain more insight into the information about the sequence and steps involved in protein folding mechanisms. In this report, a stepwise unfolding of bovine serum albumin (BSA) with guanidine hydrochloride (GuHCl) has been investigated using Raman spectroscopy in the amide I and III regions. Two-dimensional (2D) correlation analysis has been applied to reveal information on the sequential order and the dynamic properties of interaction during the unfolding process. Raman spectral signatures in the amide I region revealed that there is no significant change in secondary structures up to 2 M concentration of GuHCl. However, 2D correlation analysis further supports the observation by inferring the strengthening of secondary structure at the expense of tertiary structure. At a higher concentration of GuHCl (2–4 M), there is an accumulation of random and β-sheet structures that is mediated by small connecting segments of helices. It further accelerates the unfolding of helices and a complete collapse of structure. These analyses establish the ability of Raman spectroscopy to estimate the ensemble of secondary structures present in proteins. The results reveal the mechanistic details of unfolding, characterizing structure of intermediates even at high concentrations, and understanding the evolution of various secondary structures with respect to each other during unfolding. Such observations can be helpful in understanding the factors affecting the shape and size of proteins during folding/unfolding.

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“…Normalization of the Raman spectra is usually implemented based on the Raman band of Phenylalanine (Phe) at 1003 cm −1 because this band is not sensitive to protein conformational changes (Perevedentseva et al, 2019). Carbonyl C = O stretch near 1650 cm −1 indicated Amide I band (Logan et al, 2021) and can be deconvoluted to reveal secondary structures such as α-helix and β-pleated sheet, and so on (Kuhar & Umapathy, 2020). Peaks were marked and related to their molecular functional group in Supporting Information: Figure S3a.…”

Infrared Microspectroscopy With Multivariate Analysis to Differentiate Oral Hyperplasia From Squamous Cell Carcinoma: A Proof of Concept for Early Diagnosis

“…A linear baseline was fitted for this region. The initial position, height, and bandwidth of the bands used for fitting were determined using secondderivative spectra [36][37][38]. The second-derivative spectra were calculated using the Savitzky-Golay derivative algorithm.…”

Raman Spectra of Amino Acids and Peptides from Machine Learning Polarizabilities

“…3,4 In this case, peaks pertaining to the peptide bonds (typically amide I and II) can be used to understand the impact of folding on the Raman spectra. 5,6 Another highly promising development is the use of surface-enhanced Raman spectroscopy to study the primary structures of protein, i.e., their sequence of amino acids. As the protein moves across the plasmonic hotspot, this technique could allow one to record the spectra of individual amino acids sequentially.…”

“…Raman spectroscopy is an important tool in modern research, because it represents a nondestructive method to study the vibrational properties of both solids and molecules. In biochemistry, it can be used to study the complex structures of large molecules such as proteins or peptides. , For instance, Raman spectra contain important information on the folding of proteins. , In this case, peaks pertaining to the peptide bonds (typically amide I and II) can be used to understand the impact of folding on the Raman spectra. , Another highly promising development is the use of surface-enhanced Raman spectroscopy to study the primary structures of protein, i.e., their sequence of amino acids. As the protein moves across the plasmonic hotspot, this technique could allow one to record the spectra of individual amino acids sequentially. , In all of these cases, however, it is still necessary to have good reference spectra in order to perform correct assignment.…”