Studies of biomolecular conformations and conformational dynamics by mass spectrometry

Abstract

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I.	Introduction	38
	A. Protein Conformations and Dynamics	38
	B. Protein-Energy Landscapes: The Folding Problem and Dynamics in the Native State	38
	1. Protein Conformational Ensembles and Energy Landscapes: Enthalpic and Entropic Considerations	38
	2. Kinetic and Equilibrium Intermediates	39
	3. Definition and Specific Examples of Protein Conformations: Native Conformations, Random Coils, Late-Folding Intermediates, and Molten Globules	40
	C. Folding and Binding	41
II.	Mass Spectrometry-Based Approaches to Study Protein Conformations and Dynamics	41
	A. Positive-Ion Charge-State Distributions and Biomolecular Shapes in Solution (ESI MS)	41
	B. Amide Hydrogen Exchange	44
	1. Mechanism	44
	2. HDX Pulse Labeling	45
	3. Site-Specific Measurement of HDX by Protein Fragmentation	46
	C. Differential Chemical Labeling	49
	D. Gas-Phase Methods of Probing Multi-Protein Complex Topology	53
III.	Protein-Folding Studies	53
	A. Characterization of Kinetic Intermediates in Refolding Experiments	53
	B. Characterization of Equilibrium Intermediates in Unfolding Experiments	55
IV.	Studies of Protein-Structure and Dynamics Under Native Conditions	58
	A. Characterization of Higher-Order Structure	58
	1. Quaternary Structure	58
	2. Secondary and Tertiary Structure	60
	B. Probing Structural Dynamics Under Native Conditions	60
	C. Ligand- and Substrate-Induced Conformational Changes in Proteins	62
	V. Conclusions	65
	A. Place of MS in Experimental Biophysics: Uniqueness and Complementarity to Other Techniques	65
	B. Future Directions in the Field: Perspectives and Challenges	65
Acknowledgments		66
References		66

In the post-genomic era, a wealth of structural information has been amassed for proteins from NMR and crystallography. However, static protein structures alone are not a sufficient description: knowledge of the dynamic nature of proteins is essential to understand their wide range of functions and behavior during the life cycle from synthesis to degradation. Furthermore, few proteins have the ability to act alone in the crowded cellular environment. Assemblies of multiple proteins governed by complex signaling pathways are often required for the tasks of target recognition, binding, transport, and function. Mass spectrometry has emerged over the past several years as a powerful tool to address many of these questions. Recent improvements in “soft” ionization techniques have enabled researchers to study proteins and biomolecular complexes, both directly and indirectly. Likewise, continuous improvements in instrumental design in recent years have resulted in a dramatic expansion of the m/z range and resolution, enabling observation of large multi-protein assemblies whose structures are retained in the gas phase. In this article, we discuss some of the mass spectrometric techniques applied to investigate the nature of the conformations and dynamical properties that govern protein function. © 2002 Wiley Periodicals, Inc. Mass Spec Rev 21:37–71, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mas.10017