Structural analysis of proteins by isotope-edited FTIR spectroscopy

Abstract

Abstract. Structure determination of multidomain proteins or protein-membrane complexes is one of the most challenging tasks in modern structural biology. High-resolution techniques, like NMR or X-ray crystallography, are limited to molecules of moderate size or those that can be crystallized easily. Both methods encounter serious technical obstacles in structural analysis of protein-membrane systems. This work describes an emerging biophysical technique that combines segmental isotope labeling of proteins with Fourier transform infrared (FTIR) spectroscopy, which provides site-specific structural information on proteins and allows structural characterization of protein-membrane complexes. Labeling of a segment of the protein with 13 C results in infrared spectral resolution of the labeled and unlabeled parts and thus allows identification of structural changes in specific domains/segments of the protein that accompany functional transitions. Segmental isotope labeling also allows determination of the precise configuration of protein-membrane complexes by polarized attenuated total reflection FTIR (ATR-FTIR) spectroscopy. These new developments offer solutions to functionally important site-specific structural changes in proteins and protein-membrane complexes that are hard to approach using conventional methods.

Quercetin-3-Rutinoside Blocks the Disassembly of Cholera Toxin by Protein Disulfide Isomerase

“…This could affect the structural change in PDI that is required for disassembly of the CT holotoxin. We examined this possibility with Fourier transform-infrared (FTIR) spectroscopy, a technique that can detect changes in the secondary and tertiary structures of proteins [44,45]. As seen in Figure 4A, the spectrum of PDI (dashed line) did not shift or change shape in the presence of a 150-fold molar excess of Q3R (grey solid line).…”

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

“…Uniformly 13 C-labeled CTA1 was used to differentiate between the spectra of CTA1 and unlabeled Hsp90. An ϳ50 cm Ϫ1 shift in the spectra of the 13 C-labeled protein allows it to be resolved from the spectra of the unlabeled protein, thus facilitating analysis of the individual proteins (6,50,53). The spectra of CTA1 alone (Fig.…”

ADP‐ribosylation factor 6 acts as an allosteric activator for the folded but not disordered cholera toxin A1 polypeptide

“…Moreover, in cases when two proteins are combined in one sample, the structures of both can be determined by isotope‐edited FTIR in which one of the proteins is uniformly labelled with 13 C. 13 C labelling is a common procedure that does not alter the conformation or function of a protein. However, the heavier nuclear mass of the stable 13 C isotope generates a spectral downshift which allows the FTIR spectrum of a 13 C‐labelled protein to be resolved from the spectra of unlabelled proteins (Tatulian et al ., 2002; 2005; Tatulian, ; Taylor et al ., ). We have previously employed this approach and have reported the FTIR amide I band of 13 C‐labelled CTA1 exhibits a 45–50 cm −1 downshift in comparison to the spectrum of unlabelled CTA1.…”

“…The content of each secondary structure in the protein was determined as the area of the corresponding component relative to the total amide I area, corrected for the respective extinction coefficient (Taylor et al ., ; Tatulian, ). Since uniform 13 C‐labelling of proteins results in a 45–50 cm −1 downshift of the amide I band (Tatulian, 2010; 2013; Taylor et al ., ), the assignments of amide I components of the 13 C‐labelled CTA1 to secondary structure types were done in a similar manner, taking into account the spectral downshift. Finally, the secondary structures of combined unlabelled ARF6 and 13 C‐labelled CTA1 were estimated by dividing each amide I band fraction assigned to ARF6 or 13 C‐CTA1 by the fraction of the respective protein in the sample in terms of the number of amino acid residues, as described earlier in the Supplemental Data of Taylor et al .…”

“…Thus, the structure of the CTA1 polypeptide allows it to both exploit and evade ERAD. CTA1 enters the cytosol in an unfolded state as it passes through one or more protein-conducting channels in the ER membrane (Schmitz et al, 2000;Bernardi et al, 2008;2010;Dixit et al, 2008;Saslowsky et al, 2010). The translocated pool of CTA1 must therefore regain a folded conformation in order to act upon its Gsα target which is located in lipid rafts at the cytoplasmic face of the plasma membrane (Oh and Schnitzer, 2001;Allen et al, 2007;Kamata et al, 2008).…”