Second-Derivative IR Spectroscopy Reveals Hidden Modes of Structural Organization within Conformational Disorder

Intrinsically disordered proteins (IDPs) and regions constitute a significant fraction of the proteome. Unlike globular proteins, IDPs exist as dynamic conformational ensembles, allowing functional versatility while challenging conventional structural methods, necessitating an integrated, multi-method approach. Here, we systematically compare IDPs and globular proteins using Fourier Transform Infrared (FTIR) and circular dichroism (CD) spectroscopies to investigate not simply the presence of disorder but also its structural organization. By analyzing the second derivatives of the Amide I′ absorption spectra, we identified a distinct spectral fingerprint of IDPs. Detailed analysis reveals that the spectral features encode distinct modes of structural organization within conformationally heterogeneous ensembles. Notably, the integration of second-derivative FTIR (SD-IR) and CD spectroscopies uncovers conformational states that are not captured by conventional techniques devoted to secondary structure characterization. Specifically, we identified conditions in which proteins retain a conformational order despite exhibiting random coil CD signatures (“ordered random coils”) as well as states displaying canonical α-helical CD features while maintaining substantial structural heterogeneity (“disordered α-helices”). Molecular dynamics simulations support these assignments, revealing the structural basis underlying the spectroscopic findings. Finally, we demonstrate the efficacy of SD-IR spectroscopy for validating putative IDPs, exemplified by the silkworm protein sericin. Overall, this study establishes second-derivative analysis of the Amide I′ band not merely as a diagnostic marker of disorder but also as a powerful approach to resolve distinct organizational states within conformationally heterogeneous protein ensembles.