Biophysical techniques for protein secondary structure analysis: a comprehensive review

Adil Ahiri; Adil Ahiri

doi:10.3934/biophy.2026010

AIMS Biophysics

2026, Volume 13, Issue 2: 162-188. doi: 10.3934/biophy.2026010

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Review

Biophysical techniques for protein secondary structure analysis: a comprehensive review

Adil Ahiri ^,

Polydisciplinary Faculty of Safi, Cadi Ayyad University, Safi, Morocco

Received: 14 January 2026 Revised: 14 March 2026 Accepted: 03 April 2026 Published: 16 April 2026

The determination of protein secondary structure constitutes a fundamental step in understanding protein function, folding mechanisms, and disease pathology. This comprehensive review presents a systematic methodological comparison of the principal biophysical techniques employed for secondary structure elucidation, spanning seven decades of technological development from Pauling and Corey's foundational discoveries to contemporary high-resolution methods. We examine spectroscopic approaches, including circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy, which provide rapid global quantification of secondary structure content with minimal sample requirements, alongside high-resolution techniques, including nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and cryo-electron microscopy (cryo-EM), which offer atomic-level structural details. For each technique, we analyze theoretical foundations, experimental protocols, deconvolution algorithms, accuracy benchmarks, and optimal application domains. Comparative analysis reveals that no single technique achieves optimal performance across all secondary structure classes: FTIR generally provides improved accuracy for β-sheet-rich systems, particularly when sheet topology discrimination is required. NMR uniquely provides residue-level resolution with dynamics information across multiple timescales, whereas crystallography and cryo-EM define atomic-resolution standards for static structures. Complementary techniques, including small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and Förster resonance energy transfer (FRET), provide essential orthogonal information on global shape, protection patterns, and distance constraints. We present detailed decision frameworks for technique selection based on sample characteristics and research objectives, propose integrative multi-technique workflows, and identify critical gaps in current methodology, including the need for comprehensive multi-method benchmarking and standardized protocols for challenging protein systems such as membrane proteins and intrinsically disordered proteins. This review provides practical guidance for researchers designing experimental strategies for protein secondary structure characterization in the modern era of integrative structural biology.
- protein secondary structure,
- circular dichroism,
- FTIR spectroscopy,
- NMR spectroscopy,
- X-ray crystallography,
- cryo-electron microscopy,
- α-helix,
- β-sheet,
- protein folding
Citation: Adil Ahiri. Biophysical techniques for protein secondary structure analysis: a comprehensive review[J]. AIMS Biophysics, 2026, 13(2): 162-188. doi: 10.3934/biophy.2026010

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Abstract

The determination of protein secondary structure constitutes a fundamental step in understanding protein function, folding mechanisms, and disease pathology. This comprehensive review presents a systematic methodological comparison of the principal biophysical techniques employed for secondary structure elucidation, spanning seven decades of technological development from Pauling and Corey's foundational discoveries to contemporary high-resolution methods. We examine spectroscopic approaches, including circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy, which provide rapid global quantification of secondary structure content with minimal sample requirements, alongside high-resolution techniques, including nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and cryo-electron microscopy (cryo-EM), which offer atomic-level structural details. For each technique, we analyze theoretical foundations, experimental protocols, deconvolution algorithms, accuracy benchmarks, and optimal application domains. Comparative analysis reveals that no single technique achieves optimal performance across all secondary structure classes: FTIR generally provides improved accuracy for β-sheet-rich systems, particularly when sheet topology discrimination is required. NMR uniquely provides residue-level resolution with dynamics information across multiple timescales, whereas crystallography and cryo-EM define atomic-resolution standards for static structures. Complementary techniques, including small-angle X-ray scattering (SAXS), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and Förster resonance energy transfer (FRET), provide essential orthogonal information on global shape, protection patterns, and distance constraints. We present detailed decision frameworks for technique selection based on sample characteristics and research objectives, propose integrative multi-technique workflows, and identify critical gaps in current methodology, including the need for comprehensive multi-method benchmarking and standardized protocols for challenging protein systems such as membrane proteins and intrinsically disordered proteins. This review provides practical guidance for researchers designing experimental strategies for protein secondary structure characterization in the modern era of integrative structural biology.

Acknowledgments

The author gratefully acknowledges helpful discussions during the preparation of this manuscript.

Conflict of interest

The author declare no conflicts of interest.

Author contributions

Adil Ahiri - conceptualization, literature review, writing, editing.

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