Measuring inter-protein pairwise interaction energies from a single native mass spectrum by double-mutant cycle analysis

Sokolovski, Miri; Cvetićanin, Jelena; Hayoun, Déborah; Korobko, Ilia; Sharon, Michal; Horovitz, Amnon

doi:10.1038/s41467-017-00285-1

Cited by 29 publications

(34 citation statements)

References 18 publications

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“…5; Fersht et al, 1992). Double mutant cycle analysis has been widely used to measure the strengths of intramolecular and intermolecular side-chain interactions for both globular and membrane proteins (Serrano et al, 1990, 1991; Doura and Fleming, 2004; Hong et al, 2006, 2007; Harel et al, 2007; Joh et al, 2008; Sokolovski et al, 2017). To minimize the possibility of creating new interactions after mutation, we replaced each active site residue with alanine (Horovitz, 1996).…”

Section: Resultsmentioning

confidence: 99%

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network

Gaffney

2018

J. Gen. Physiol.

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Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔGInter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

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Section: Resultsmentioning

confidence: 99%

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network

Gaffney

2018

J. Gen. Physiol.

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“…A very interesting application of double mutant cycles has been recently implemented by studying the formation of protein-protein interactions via native mass spectrometry [ 51 ]. The intrinsic power of this technique lies in the ability to transfer protein complexes to the gas phase, without altering their native state [ 52 , 53 , 54 ].…”

Section: Double Mutant Cycles By Native Mass Spectrometrymentioning

confidence: 99%

“…Double mutant cycles by mass spectrometry have been successfully employed to study the binding between the proteins E9 and Im2 [ 51 ] and, more recently, to understand the dimerization of SOD1 in naturally-occurring mutants associated with ALS. The authors analyzed the coupling constants by co-expressing wild-type SOD1 with ALS-causing mutations [ 57 ].…”

Section: Double Mutant Cycles By Native Mass Spectrometrymentioning

confidence: 99%

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

Pagano

Toto

Malagrinò

et al. 2021

IJMS

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Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted ‘energetic coupling’ describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein–ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.

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“… 81 An earlier study has shown that pairwise interaction energies can be determined from a single mass spectrum. 82 On the basis of this understanding, four different proteins, consisting of the two wild type (wt) and two mutated (mut) variants, were coexpressed in the same bacterial cells. 21 The spectrum measured from the crude lysate showed the four coexisting complexes (wt/wt, wt/mut, mut/wt, and mut/mut), from which the coupling energy was calculated, obviating the need to purify each of the four proteins and determine individual binding constants for each of the generated dimers.…”

Section: Direct Msmentioning

confidence: 99%

Mass Spectrometry Analysis of Intact Proteins from Crude Samples

2020

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Analysis of intact proteins by native mass spectrometry has emerged as a powerful tool for obtaining insight into subunit diversity, post-translational modifications, stoichiometry, structural arrangement, stability, and overall architecture. Typically, such an analysis is performed following protein purification procedures, which are time consuming, costly, and labor intensive. As this technology continues to move forward, advances in sample handling and instrumentation have enabled the investigation of intact proteins in situ and in crude samples, offering rapid analysis and improved conservation of the biological context. This emerging field, which involves various ion source platforms such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) for both spatial imaging and solution-based analysis, is expected to impact many scientific fields, including biotechnology, pharmaceuticals, and clinical sciences. In this Perspective, we discuss the information that can be retrieved by such experiments as well as the current advantages and technical challenges associated with the different sampling strategies. Furthermore, we present future directions of these MS-based methods, including current limitations and efforts that should be made to make these approaches more accessible. Considering the vast progress we have witnessed in recent years, we anticipate that the advent of further innovations enabling minimal handling of MS samples will make this field more robust, user friendly, and widespread.

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Measuring inter-protein pairwise interaction energies from a single native mass spectrum by double-mutant cycle analysis

Cited by 29 publications

References 18 publications

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

Mass Spectrometry Analysis of Intact Proteins from Crude Samples

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