Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry

Mass spectrometry with chemical cross-linking was used to probe the conformational changes of HSA (human serum albumin) in solution on interaction with monounsaturated OA (oleic acid) or polyunsaturated AA (arachidonic acid) or DHA (docosahexaenoic acid). Fatty acid-free or -bound HSA was modified with lysine-specific cross-linkers and digested with trypsin. Cross-linked peptides were analysed by nano-electrospray ionization MS to localize the sites of cross-linking. Our data indicated that a local conformational change involving movement of the side chains of Lys-402 of subdomain IIIA or Lys-541 of subdomain IIIB occurred upon binding of all three fatty acids. Our data also indicated that the side chains of Lys-205 (IIA) and Lys-466 (IIIA) moved closer towards each other upon binding AA or DHA, but not OA, suggesting that the conformations of HSA when bound to mono- and poly-unsaturated fatty acids are distinctively different. While these observations agreed with previous X-ray crystallographic studies, the distances between ε-amino groups of most cross-linked lysine pairs were shorter than the crystal structure predicted, possibly reflecting a discrepancy between the solution and crystal structures. This method can serve as a useful complement to X-ray crystallography, particularly in probing the structure of a protein in solution.

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Monitoring Conformational Changes in Protein Complexes Using Chemical Cross-Linking and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: The Effect of Calcium Binding on the Calmodulin—Melittin Complex

European Journal of Mass Spectrometry ◽

10.1255/ejms.882 ◽

2007 ◽

Vol 13 (4) ◽

pp. 281-290 ◽

Cited By ~ 8

Author(s):

Petr Novak ◽

Vladimir Havlicek ◽

Peter J. Derrick ◽

Kyle A. Beran ◽

Sajid Bashir ◽

...

Keyword(s):

Mass Spectrometry ◽

Fourier Transform ◽

Conformational Changes ◽

Calcium Binding ◽

Cyclotron Resonance ◽

Structural Changes ◽

Cross Linking ◽

Ion Cyclotron Resonance ◽

Ion Cyclotron ◽

Chemical Cross Linking

Calmodulin is an EF hand calcium binding protein. Its binding affinities to various protein/peptide targets often depend on the conformational changes induced by the binding of calcium. One such target is melittin, which binds tightly to calmodulin in the presence of calcium, and inhibits its function. Chemical cross-linking combined with Fourier transform ion cyclotron resonance mass spectrometry has been employed to investigate the coordination of calmodulin and melittin in the complex at different concentrations of calcium. This methodology can be used to monitor structural changes in proteins induced by ligand binding and to study the effects these changes have on non-covalent interactions between proteins. Cross-linking results indicate that the binding place of the first melittin in the calcium-free calmodulin form is the same as in the calcium-loaded calmodulin/melittin complex.

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Effects of Ethanol on Conformational Changes of Akt Studied by Chemical Cross-Linking, Mass Spectrometry, and 18O Labeling

ACS Chemical Biology ◽

10.1021/cb2003237 ◽

2011 ◽

Vol 7 (2) ◽

pp. 387-394 ◽

Cited By ~ 7

Author(s):

Bill X. Huang ◽

Hee-Yong Kim

Keyword(s):

Mass Spectrometry ◽

Conformational Changes ◽

Cross Linking ◽

18O Labeling ◽

Chemical Cross Linking

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Characterization of an Antagonist Interleukin-6 Dimer by Stable Isotope Labeling, Cross-linking, and Mass Spectrometry

Journal of Biological Chemistry ◽

10.1074/jbc.m207370200 ◽

2002 ◽

Vol 277 (48) ◽

pp. 46487-46492 ◽

Cited By ~ 75

Author(s):

Thomas Taverner ◽

Nathan E. Hall ◽

Richard A. J. O'Hair ◽

Richard J. Simpson

Keyword(s):

Mass Spectrometry ◽

Stable Isotope ◽

Interleukin 6 ◽

Isotope Labeling ◽

Stable Isotope Labeling ◽

Cross Linking

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A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

10.1101/2020.11.05.369280 ◽

2020 ◽

Author(s):

Jian-Hua Wang ◽

Yu-Liang Tang ◽

Rohit Jain ◽

Fan Xiao ◽

Zhou Gong ◽

...

Keyword(s):

Mass Spectrometry ◽

Conformational Changes ◽

Protein Interactions ◽

Guanidine Hydrochloride ◽

Rnase A ◽

Mass Spectrometry Analysis ◽

Cross Linking ◽

Protein Protein Interactions ◽

Protein Structure Analysis ◽

Cross Linker

AbstractChemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.

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Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry

Journal of Biological Chemistry ◽

10.1074/jbc.m808521200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16354-16368 ◽

Cited By ~ 13

Author(s):

Martijn W. H. Pinkse ◽

Dirk T. S. Rijkers ◽

Wolfgang R. Dostmann ◽

Albert J. R. Heck

Keyword(s):

Mass Spectrometry ◽

Protein Kinase ◽

Isotope Labeling ◽

Cross Linking ◽

Specific Substrate ◽

Dimerization Domain ◽

Dependent Protein Kinase ◽

Catalytic Core ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

The inhibitor peptide DT-2 (YGRKKRRQRRRPPLRKKKKKH) is the most potent and selective inhibitor of the cGMP-dependent protein kinase (PKG) known today. DT-2 is a construct of a PKG tight binding sequence (W45, LRKKKKKH, KI = 0.8 μm) and a membrane translocating sequence (DT-6, YGRKKRRQRRRPP, KI = 1.1 μm), that combined strongly inhibits PKG catalyzed phosphorylation (KI = 12.5 nm) with ∼1000-fold selectivity toward PKG over protein kinase A, the closest relative of PKG. However, the molecular mechanism behind this inhibition is not entirely understood. Using a combination of photoaffinity labeling, stable isotope labeling, and mass spectrometry, we have located the binding sites of PKG-specific substrate and inhibitor peptides. Covalent linkage of a PKG-specific substrate analogue was localized in the catalytic core on residues 356–372, also known as the glycine-rich loop, essential for ATP binding. By analogy, the individual inhibitor peptides W45 and DT-6 were also found to cross-link near the glycine-rich loop, suggesting these are both substrate competitive inhibitors. A bifunctional photoreactive analogue of DT-2 was found to generate dimers of PKG. This cross-linking induced covalent PKG dimerization was not observed for an N-terminal deletion mutant of PKG, which lacks the dimerization domain. In addition, non-covalent mass spectrometry was used to determine binding stoichiometry and binding order of the inhibitor peptides. Dimeric PKG binds two W45 and DT-6 peptides, whereas only one DT-2 molecule was observed to bind to the dimeric PKG. Taken together, these findings imply that (i) the two individual components making up DT-2 are both targeted against the substrate-binding site and (ii) binding of a single DT-2 molecule inactivates both PKG monomers simultaneously, which is an indication that (iii) in cGMP-activated PKG the catalytic centers of both subunits may be in each other's proximity.

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Comparative cross-linking and mass spectrometry of an intact F-type ATPase suggest a role for phosphorylation

Nature Communications ◽

10.1038/ncomms2985 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 102

Author(s):

Carla Schmidt ◽

Min Zhou ◽

Hazel Marriott ◽

Nina Morgner ◽

Argyris Politis ◽

...

Keyword(s):

Mass Spectrometry ◽

Conformational Changes ◽

Mass Spectra ◽

Nucleotide Binding ◽

Cross Linking ◽

Spinach Chloroplasts ◽

Cross Links ◽

Chemical Cross Linking

Abstract F-type ATPases are highly conserved enzymes used primarily for the synthesis of ATP. Here we apply mass spectrometry to the F1FO-ATPase, isolated from spinach chloroplasts, and uncover multiple modifications in soluble and membrane subunits. Mass spectra of the intact ATPase define a stable lipid ‘plug’ in the FO complex and reveal the stoichiometry of nucleotide binding in the F1 head. Comparing complexes formed in solution from an untreated ATPase with one incubated with a phosphatase reveals that the dephosphorylated enzyme has reduced nucleotide occupancy and decreased stability. By contrasting chemical cross-linking of untreated and dephosphorylated forms we show that cross-links are retained between the head and base, but are significantly reduced in the head, stators and stalk. Conformational changes at the catalytic interface, evidenced by changes in cross-linking, provide a rationale for reduced nucleotide occupancy and highlight a role for phosphorylation in regulating nucleotide binding and stability of the chloroplast ATPase.

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