**2. Results**

The NUV-CD spectra of the folded and unfolded states of IgG1 and IgG2 are shown in Figure 1. The effects of HOS on the differential absorption of left and right circularly polarized light can be seen in the spectral comparisons of the folded and unfolded states of IgG1 and IgG2, in Figure 1A,B, respectively. In general, the NUV-CD spectra of native proteins are characterized by distinct features at around 293 and 286 nm attributable to tryptophan, at 285 to 270 nm attributable to tyrosine and tryptophan, and 250–265 nm attributable to phenylalanine, superimposed over the disulfide signal from 250 to 280 nm. While the unfolded spectra of both IgG's show relatively featureless lines close to zero (Figure 1D), the folded spectra show absorption changes for the chromophores: tryptophan, tyrosine, and phenylalanine, indicating that these pendent groups are incorporated into highly organized portions of the protein, i.e., tertiary structure. Furthermore, even small differences in HOS and primary structure give rise to unique spectra for the folded states of the two mAbs, allowing them to be distinguished from each other as well (Figure 1C).

**Figure 1.** NUV-CD spectra of the folded and unfolded samples of IgG1 (**A**) and IgG2 (**B**). Comparison of the spectra from the folded states of the IgG1 and IgG2 molecules in (**C**), and the unfolded states for these two molecules in (**D**).

The FLD spectra of the folded and unfolded states of IgG1 and IgG2 are shown in Figure 2. The emission wavelengths of the internal fluorophores: tryptophan, phenylalanine, and tyrosine, are sensitive to the polarity of their environments. Higher polarity environments, particularly water from the solvent, cause the wavelengths of emission to lengthen (i.e., red shift). Therefore, unfolded proteins with more solvent-exposed fluorophores will appear more red-shifted than proteins whose tertiary structure tends to sequester these fluorophores in internal, more non-polar environments (Figure 2A,B) [10]. In our study, for both the mAbs IgG1 and IgG2, the folded spectra have a peak around 323 nm, and upon unfolding, the peak shifts to around 345 nm. We also observe that the fluorescence intensity upon unfolding increases (by almost 30%) for both mAbs and is due to the fact that the fluorescence quenching groups are further apart in the unfolded protein than in the native protein, resulting in significant lowering of energy transfer efficiency in the native protein [11]. However, little else about the HOS of the mAbs can be seen by their essentially indistinguishable folded spectra (Figure 2C). In addition, as clearly indicated in Figure 2D, the unfolded spectra for the two mAbs are essentially identical.

**Figure 2.** FLD spectra of the folded and unfolded samples of IgG1 (**A**) and IgG2 (**B**). Comparison of the spectra from the folded states of the IgG1 and IgG2 molecules in (**C**), and the unfolded states for these two molecules in (**D**).

The 1H-13C HSQC NMR methyl spectra of the folded and unfolded states of IgG1 and IgG2 are shown in Figure 3. NMR relays information about the local magnetic environments of the nuclei under investigation both through chemical bonds and spatially by the other atoms surrounding them. Atoms in more magnetically shielded environments have lower chemical shifts (plotted in ppms), while atoms in less shielded environments have higher chemical shifts. 2D 1H-13C HSQC NMR experiments are designed to correlate both protons (*x*-axis) and the carbon-13 atoms (*y*-axis) that they are directly attached to in a molecule. Since proteins are almost entirely composed of protons and carbons, NMR provides a wealth of information about primary structure and all levels of HOS. The resolution of primary structure can be seen in the unfolded spectra of IgG1 and IgG2 (Figure 3B,D). In the absence of any ordered secondary or tertiary structure, the unique chemical signals of different amino acids are resolved and by comparison to reported random-coil (unfolded) chemical shifts, side-chain units, particularly the methyl groups, can be tentatively assigned (Figure 4) [12]. In the folded state, the various magnetic environments of each individual amino acid disperse the side-chain signals to produce a truly unique spectrum for each protein, which is dependent upon all levels of HOS, as shown in Figure 3A,C.

**Figure 3.** 2D 1H-13C HSQC spectra of the folded and unfolded samples of IgG1 and IgG2. (**A**,**B**) show IgG1 in the folded and unfolded state respectively, whereas (**C**,**D**) show IgG2 in the folded and unfolded states respectively. 1H is represented on the x axis (f2), and 13C is represented on the y axis (f1).

**Figure 4.** Multiplicity-edited 2D 1H-13C HSQC spectra of the unfolded state of IgG2. Red peaks are either CH or CH3, blue peaks are CH2. The methyl groups of each amino acid are shown in red boxes corresponding to the random coil 1H and 13C shift ranges for the six methyl groups [12]. 1H is represented on the x axis (f2), and 13C is represented on the y axis (f1).
