Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry

Engen, John R.; Wales, Thomas E.; Chen, Shugui; Marzluff, Elaine M.; Hassell, Kerry M.; Weis, David D.; Smithgall, Thomas E.

doi:10.1080/0144235x.2012.751175

Cited by 38 publications

(57 citation statements)

References 123 publications

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“…Taken together, secondary structure is not the only factor that contributes to SH3 domain unfolding. As previously speculated, 14 tertiary structures driven by side-chains must play a role in stability, although we are unable to ascertain exactly how. HX MS has been used to assess SH3 domain interactions with potential ligands as changes in EX1 kinetics.…”

Section: 38mentioning

confidence: 77%

“…Both of these domains were fully deuterated after 10 seconds of labeling, consistent with a lack of stable structure under these conditions. For the sake of comparison, the previous studies 14,26 involving cooperative unfolding in other SH3 domains indicate that Hck SH3 and a-spectrin undergo the greatest documented increases in peak width for an SH3 domain (measured at FWHM) upon deuteration of 15.4 and 19.1 Da, respectively.…”

Section: Sequence Comparison Of Src and Tec-family Kinase Sh3 Domainsmentioning

confidence: 99%

“…However, the structural mechanism responsible for this unfolding event is not clear. 14 We prepared a phylogenetic tree based on the sequences of all SH3 domains studied by HX MS to date, including the Tec-family kinase SH3 domains investigated here (Fig. 6).…”

Section: 38mentioning

confidence: 99%

“…11 Previously, our group probed the solution dynamics of a number of SH3 domains with hydrogen exchange mass spectrometry (HX MS) and found unpredictable dynamics and partial unfolding at near-physiological conditions. 5,[12][13][14] In one study, the dynamics of the SH3 domains of eight members of the Src-family of non-receptor tyrosine kinases (nRTK) along with four other biologically relevant SH3 domains were probed side-by-side. 13 This small sample of SH3 domains displayed a surprisingly wide range of dynamic properties, which was not expected given both their sequence and structural homology, particularly among the members from the Src-family kinases.…”

Section: Introductionmentioning

confidence: 99%

“…HIV-1 Nef binding substantially slows the unfolding half-life of the Hck SH3 domain, 5,14 an event that correlates with Nef:Hck complex formation and kinase activation. Nef-dependent activation of Hck contributes to HIV-1 replication, in that selective inhibitors of this pathway interfere with viral replication in vitro.…”

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confidence: 99%

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Dynamics of the Tec‐family tyrosine kinase SH3 domains

et al. 2016

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The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X-ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src-family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src-family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher-order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra-and intermolecular binding assays of proteins containing the domains.

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Section: 38mentioning

confidence: 77%

Section: Sequence Comparison Of Src and Tec-family Kinase Sh3 Domainsmentioning

confidence: 99%

Section: 38mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Dynamics of the Tec‐family tyrosine kinase SH3 domains

et al. 2016

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Dissecting Cooperative Communications in a Protein with a High‐Throughput Single‐Molecule Scalpel

Cui

Sangeetha

et al. 2014

ChemPhysChem

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Miscued communication often leads to misfolding and aggregation of the proteins involved in many diseases. Owing to the ensemble average property of conventional techniques, detailed communication diagrams are difficult to obtain. Mechanical unfolding affords an unprecedented perspective on cooperative transitions by observing a protein along a trajectory defined by two mutated cysteine residues. Nevertheless, this approach requires tedious sample preparation at the risk of altering native protein conformations. To address these issues, we applied click chemistry to tether a protein to the two dsDNA handles through primary amines in lysine residues as well as at the N terminus. As a proof of concept, we used laser tweezers to mechanically unfold and refold calmodulin along 36 trajectories, maximally allowed by this strategy in a single batch of protein preparation. Without a priori knowledge of the particular residues to which the double-stranded DNA handles attach, we used hierarchical cluster analysis to identify 20 major trajectories, according to the size and the pattern of unfolding transitions. We dissected the cooperativity into all-or-none and partially cooperative events, which represent strong and weak high-order interactions in proteins, respectively. Although the overall cooperativity is higher within the N or C lobe than that between the lobes, the all-or-none cooperativity is anisotropic among different the unfolding trajectories and becomes relatively more predominant when the size of the protein segments increases. The average cooperativity for all-or-none transitions falls within the expected range observed by ensemble techniques, which supports the hypothesis that unfolding of a free protein can be reconstituted from individual trajectories.

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Lysine residues in the N-terminal huntingtin amphipathicα-helix play a key role in peptide aggregation

et al. 2015

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Huntington's disease is a genetic neurodegenerative disorder caused by an expansion in a polyglutamine domain near the N-terminus of the huntingtin (htt) protein that results in the formation of protein aggregates. Here, htt aggregate structure has been examined using hydrogen-deuterium exchange techniques coupled with tandem mass spectrometry. The focus of the study is on the 17-residue N-terminal flanking region of the peptide that has been shown to alter htt aggregation kinetics and morphology. A top-down sequencing strategy employing electron transfer dissociation is utilized to determine the location of accessible and protected hydrogens. In these experiments, peptides aggregate in a deuterium-rich solvent at neutral pH and are subsequently subjected to deuterium-hydrogen back-exchange followed by rapid quenching, disaggregation, and tandem mass spectrometry analysis. Electrospray ionization of the peptide solution produces the [M + 5H](5+) to [M + 10H](10+) charge states and reveals the presence of multiple peptide sequences differing by single glutamine residues. The [M + 7H](7+) to [M + 9](9+) charge states corresponding to the full peptide are used in the electron transfer dissociation analyses. Evidence for protected residues is observed in the 17-residue N-terminal tract and specifically points to lysine residues as potentially playing a significant role in htt aggregation.

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Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry

Cited by 38 publications

References 123 publications

Dynamics of the Tec‐family tyrosine kinase SH3 domains

Dynamics of the Tec‐family tyrosine kinase SH3 domains

Dissecting Cooperative Communications in a Protein with a High‐Throughput Single‐Molecule Scalpel

Lysine residues in the N-terminal huntingtin amphipathicα-helix play a key role in peptide aggregation

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