[15] Thermodynamic parameters from hydrogen exchange measurements

Bai, Yawen; Englander, Joan J.; Mayne, Leland; Milne, John; Englander, S. Walter

doi:10.1016/0076-6879(95)59051-x

Cited by 110 publications

(111 citation statements)

References 18 publications

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“…For the ⌬G opening computation, we considered that unfolding of the R-subunit has been previously adequately described by using a two-state model (14) and that at pH 6.5 an EX2 Linderstrøm-Lang mechanism generally applies (36,38). As a result, the average PF for residues with logPF cAMP-Bound Ͼ 6 (i.e., 6.6 Ϯ 0.5) leads to a ⌬G opening of ϷRTln(10 6.6 Ϯ 0.5 ) ϭ 9.2 Ϯ 0.7 kcal/mol.…”

Section: H/d Exchange Of the ␤-Subdomain Inner Core Amides Is Concertmentioning

confidence: 99%

cAMP activation of PKA defines an ancient signaling mechanism

Das

Esposito

Abu-Abed

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

115

106

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allostery ͉ NMR ͉ cyclic nucleotide binding domain T he cAMP binding domain (CBD) and cAMP are conserved from bacteria to humans as a ubiquitous signaling mechanism to translate extracellular stress signals into appropriate biological responses (1). The major receptor for cAMP in higher eukaryotes, cAMP-dependent PKA (2), is ubiquitous in mammalian cells where it exists in two forms: the inactive tetrameric holoenzyme and the active dissociated catalytic subunit (Csubunit). In the inactive holoenzyme, two C-subunits are bound to a dimeric regulatory subunit (R-subunit) (Fig. 1a). Upon binding cAMP, the R-subunits undergo a conformational change that unleashes the active C-subunits (3, 4). The Rsubunits are composed of an N-terminal dimerization/docking domain, a flexible linker that includes an autoinhibitory segment, and two tandem CBDs (CBD-A and CBD-B; Fig. 1b) (5). The CBD-A of the isoform I␣ of the R-subunit of PKA (RI␣) contains a noncontiguous ␣-subdomain, which mediates the interactions with the C-subunit and a contiguous ␤-subdomain that forms a ␤-sandwich and contains the cAMP binding pocket (i.e., the phosphate binding cassette or PBC) ( Fig. 1 c and d) (6).Crystal structures of CBD-A of RI␣ in its cAMP-(6) and C-bound (7) states have revealed two very different conformations, highlighting the conformational plasticity of this ancient domain. Although these static crystal structures define two stable end points, questions remain about the allosteric control of the reversible shuttling between the two states. How does the signal generated by cAMP binding to the PBC (Fig. 1 c and d) propagate through a long-range allosteric network that spans both ␣-and ␤-subdomains? Previous analyses (8-16) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA. To comprehensively understand this allosteric mechanism, it is therefore necessary to elucidate at high resolution how cAMP remodels the free energy landscape of CBD-A, which serves as the central controlling unit of PKA. For this purpose, we have investigated by NMR RI␣ (residues 119-244), a construct that spans both ␣-and ␤-subdomains of CBD-A and retains high-

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Section: H/d Exchange Of the ␤-Subdomain Inner Core Amides Is Concertmentioning

confidence: 99%

cAMP activation of PKA defines an ancient signaling mechanism

Das

Esposito

Abu-Abed

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

115

106

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“…*Present address: Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT. (Hvidt & Nielsen 1966;Woodward et al, 1982;Baldwin, 1993;Bai et al, 1995).…”

mentioning

confidence: 99%

Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR

et al. 1997

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The extent of deuterium labeling of hen lysozyme, its three-disulfide derivative, and the homologous a-lactalbumins, has been measured by both mass spectrometry and NMR. Different conformational states of the proteins were produced by varying the solution conditions. Alternate protein conformers were found to contain different numbers of ' H atoms. Furthermore, measurement in the gas phase of the mass spectrometer or directly in solution by NMR gave consistent results. The unique ability of mass spectrometry to distinguish distributions of ' H atoms in protein molecules is exemplified using samples prepared to contain different populations of 'H-labeled protein. A comparison of the peak widths of bovine a-lactalbumin in alternate solution conformations but containing the same average number of ' H atoms showed dramatic differences due to different ' H distributions in the two protein conformers. Measurement of ' H distributions by ESI-MS enabled characterization of conformational averaging and structural heterogeneity. In addition, a time course for hydrogen exchange was examined and the variation in distributions of ' H atom compared with simulations for different hydrogen exchange models. The results clearly show that exchange from the native state of bovine a-lactalbumin at 15 "C is dominated by local unfolding events.

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“…HX, which measures the exchange of backbone amide protons with the solvent, allows us to monitor the average state of individual residues within a protein. [18][19][20][21] Backbone amide protons that participate in H-bonding, or are shielded from solvent, are protected from exchange with solvent to a much greater extent than are solvent-exposed, non-H-bonded amide protons. As a result, HX experiments reveal extremely rare unfolding events, 22 which facilitate hydrogen exchange, within the nominally folded state of a protein, thus providing a means by which to characterize the excursions of TPR proteins into partially folded conformations.…”

Section: Introductionmentioning

confidence: 99%

Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

Cortajarena¹,

Mochrie²,

Regan³

2008

Journal of Molecular Biology

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Repeat proteins contain tandem arrays of a simple structural motif. In contrast to globular proteins, repeat proteins are stabilized only by interactions between residues that are relatively close together in the sequence, with no "long-range" interactions. Our work focuses on the tetratricopeptide repeat (TPR), a 34 amino acid helix-turn-helix motif found in tandem arrays in many natural proteins. Earlier, we reported the design and characterization of a series of consensus TPR (CTPR) proteins, which are built as arrays of multiple tandem copies of a 34 amino acid consensus sequence. Here, we present the results of extensive hydrogen exchange (HX) studies of the folding-unfolding behavior of two CTPR proteins (CTPR2 and CTPR3). We used HX to detect and characterize partially folded species that are populated at low frequency in the nominally folded state. We show that for both proteins the equilibrium folding-unfolding transition is nontwo-state, but sequential, with the outermost helices showing a significantly higher probability than inner helices of being unfolded. We show that the experimentally observed unfolding behavior is consistent with the predictions of a simple Ising model, in which individual helices are treated as "spin-equivalents". The results that we present have general implications for our understanding of the thermodynamic properties of repeat proteins.

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[15] Thermodynamic parameters from hydrogen exchange measurements

Cited by 110 publications

References 18 publications

cAMP activation of PKA defines an ancient signaling mechanism

cAMP activation of PKA defines an ancient signaling mechanism

Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR

Mapping the Energy Landscape of Repeat Proteins using NMR-detected Hydrogen Exchange

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