Dissecting the mechanism of Epac activation via hydrogen-deuterium exchange FT-IR and structural modeling

Exchange proteins directly activated by cAMP (Epac) make up a family of cAMP binding domain-containing proteins that play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Ras-proximate proteins. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and structural modeling. Our studies show that unlike that of cAMP-dependent protein kinase (PKA), the classic intracellular cAMP receptor, binding of cAMP to Epac does not induce significant changes in overall secondary structure and structural dynamics, as measured by FT-IR and the rate of H-D exchange, respectively. These results suggest that Epac activation does not involve significant changes in the amount of exposed surface areas as in the case of PKA activation, and conformational changes induced by cAMP in Epac are most likely confined to small local regions. Homology modeling and comparative structural analyses of the CBDs of Epac and PKA lead us to propose a model of Epac activation. On the basis of our model, Epac activation by cAMP employs the same underlying structural principal utilized by PKA, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different. In addition, we predict that during Epac activation the first beta-strand of the switchboard switches its conformation to a alpha-helix, which folds back to the beta-barrel core of the CBD and interacts directly with cAMP to form the base of the cAMP-binding pocket.     

On the mechanism of bacteriorhodopsin solubilization by surfactants

Purple membrane bacteriorhodopsin can be easily solubilized by Triton X-100 and other detergents, but not by deoxycholate. In order to understand this behavior, we have examined the effects of a variety of surfactants. We show that detergents containing the cholane ring (cholate, taurocholate, 3[(3-cholamidopropyl)diethyl-ammonio]propanesulfonic acid...) are virtually unable to solubilize native bacteriorhodopsin. However, when the protein is reconstituted in dimyristoyl phosphatidylcholine and solubilization is assayed at a temperature such that bacteriorhodopsin is in the form of monomers, solubilization by cholane detergents does occur. We propose that steric factors prevent access of the rigid planar surfactant molecules to the hydrophobic protein regions. These are perhaps located in the monomer-monomer interface, whose solvation by surfactants is essential for solubilization to occur. We note that the capacity of some detergents to solubilize bacteriorhodopsin is always associated within the same range of surfactant concentrations with bleaching (partial or total) of the protein chromophore. The detergent-induced bleaching is at least partially reversible, suggesting that free retinal remains associated to some membrane components. While some surfactant molecules remain tightly bound to the membrane protein, cholane detergents can be completely removed from bacteriorhodopsin. Our results indicate that a structure-function relationship exists for detergents applied to the solubilization of bacteriorhodopsin.     

Dynamics of hydrogen-deuterium exchange in Chlamydomonas centrin

Chlamydomonas reinhardtii centrin is a 169-amino acid residue calcium binding protein belonging to the EF-hand protein superfamily. Centrin is associated with the microtubule organizing center (MTOC) in all eukaryotes, and in Chlamydomonas, centrin is a component of the flagellar basal body apparatus. Recombinant full-length centrin, calmodulin, and terminal domain fragments [Ccen-N (residues 1-94) and Ccen-C (residues 99-169)] were used to examine hydrogen-deuterium (H --> D) exchange dynamics using combined attenuated total reflectance (ATR) Fourier transform-infrared (FT-IR) spectroscopy, curve fit, and two-dimensional correlation analysis. Analysis of the Ccen-N and Ccen-C fragments allowed separation of domain specific solvent exchange events and together with analysis of the full-length proteins provides novel insight into domain accessibility to the aqueous environment and the internal dynamics of the protein.     

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme.

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl^?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl^? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D₂O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k₂, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.     

Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.     

Kinetics of hydrogen-deuterium exchange in ATPase from a thermophilic bacterium PS3.

The kinetics of the hydrogen-deuterium exchange reaction in a stable ATPase (TF₁) from a thermophilic bacterium PS3 was followed by infrared absorption measurements. The rates of the hydrogen-deuterium exchange reactions decreased in following order; free form, TF₁·ADP, TF₁·ATP and TF₁·AMP-P(NH)P. TF₁ does not dissociate into subunits even in the absence of nucleotides, thus differences in exchange likely reflect differences in conformations of subunits. These results indicate that the structure is most restricted when ATP or AMP-P(NH)P is bound to the enzyme.     

pH and urea dependence of amide hydrogen-deuterium exchange rates in the beta-trefoil protein hisactophilin.

Amide hydrogen/deuterium exchange rates were measured as a function of pH and urea for 37 slowly exchanging amides in the beta-trefoil protein hisactophilin. The rank order of exchange rates is generally maintained under different solution conditions, and trends in the pH and urea dependence of exchange rates are correlated with the rank order of exchange rates. The observed trends are consistent with the expected behavior for exchange of different amides via global and/or local unfolding. Analysis of the pH dependence of exchange in terms of rate constants for structural opening and closing reveals a wide range of rates in different parts of the hisactophilin structure. The slowest exchanging amides have the slowest opening and closing rates. Many of the slowest exchanging amides are located in trefoil 2, but there are also some slow exchanging amides in trefoils 1 and 3. Slow exchangers tend to be near the interface between the beta-barrel and the beta-hairpin triplet portions of this single-domain structure. The pattern of exchange behaviour in hisactophilin is similar to that observed previously in interleukin-1 beta, indicating that exchange properties may be conserved among beta-trefoil proteins. Comparisons of opening and closing rates in hisactophilin with rates obtained for other proteins reveal clear trends for opening rates; however, trends in closing rates are less apparent, perhaps due to inaccuracies in the values used for intrinsic exchange rates in the data fitting. On the basis of the pH and urea dependence of exchange rates and optical measurements of stability and folding, EX2 is the main exchange mechanism in hisactophilin, but there is also evidence for varying levels of EX1 exchange at low and high pH and high urea concentrations. Equilibrium intermediates in which subglobal portions of structure are cooperatively disrupted are not apparent from analysis of the urea dependence of exchange rates. There is, however, a strong correlation between the Gibbs free energy of opening and the denaturant dependence of opening for all amides, which suggests exchange from a continuum of states with different levels of structure. Intermediates are not very prominent either in equilibrium exchange experiments or in quenched-flow kinetic studies; hence, hisactophilin may not form partially folded states as readily as IL-1 beta and other beta-trefoil proteins.     

Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry.

An exceedingly simple and convenient method is described for measuring the hydrogen-deuterium exchange behavior of peptide bond-containing molecules by ultraviolet spectrophotometry. The exchange reaction is initiated by diluting a sample from H₂O into D₂O, or the reverse, and can be followed by an easily observable optical density change in the region of peptide absorbance. The method, unlike infrared and magnetic resonance approaches, requires only small amounts of material and, unlike the tritium-Sephadex method, is not restricted to the study of large molecules. Calibrations are provided for exchange rate as a function of pD and temperature and for the change in absorbance per mole peptide group. With this information, the exchange curve to be expected for any peptide group exposed to solvent can be predicted. Comparison with the measured data can then identify peptide-group hydrogen bonding and can also give a measure of the stability of the hydrogen-bonded structure.     

Photoactivation of rhodopsin causes an increased hydrogen-deuterium exchange of buried peptide groups.

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II' bands. When rhodopsin films are exposed to D2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D2O exhibits a new phase of H/D exchange which at 10 degrees C consists of fast (time constant approximately 30 min) and slow (approximately 11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.     

On the mechanism of sodium-proton exchange in crayfish

In salt depleted crayfish net sodium and proton fluxes were coupled 1:1 as required by the frog skin-turtle bladder model. In addition, three proton pump inhibitors produced equal reductions of both fluxes. It is concluded that the model operates in these animals. Net Na+ and H+ fluxes were very small in tap water adapted animals, but regression analysis clearly showed that they were coupled, though perhaps not 1:1. Proton pump inhibitors, at concentrations that suppressed fluxes in salt-depleted crayfish, had no measureable effect on proton movement in tap water-adapted animals. Two of them (dicyclocarbodiimide and N-ethyl maleimide), caused a small reduction in Na+ influx without affecting proton efflux. These experiments provide no support for operation of the frog-turtle system in adult crayfish adapted to tap water. A 2Na+-H+ exchanger is considered from an energetic point of view. Such a system might be able to couple Na+ and H+ fluxes in dilute, near neutral solutions ([Na+] approximately 1-2 mM; pH 7).     

Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange

To characterize the intersubunit interactions underlying assembly and maturation in HIV-1, we determined the amide hydrogen exchange protection pattern of capsid protein in the immature virion and the mature virion using mass spectrometry. Alterations in protection upon maturation provide evidence for the maturation-induced formation of an interaction between the N- and C-terminal domains in half of the capsid molecules, indicating that only half of the capsid protein is assembled into the conical core.     

A hydrogen-deuterium exchange study of the amide protons of polymyxin B by nuclear-magnetic-resonance spectroscopy.

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and gamma-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two beta turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.     

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations.     

Facile hydrogen-deuterium exchange at the 5'-position of an analogue of S-adenosyl-l-methionine

S-3',4'-anhydroadenosyl-l-methionine is an analogue of the S-adenosyl-l-methionine coenzyme. Here we report on a rapid solvent exchange of the methylene protons at the 5'-position of this analogue. The rate of H/D exchange was measured by nuclear magnetic resonance spectroscopy under buffered conditions in deuterium oxide. The reaction is specific base catalyzed and displays a second-order rate constant of 2 x 10(4) M(-1) s(-1), which corresponds to a rate enhancement of 10(12) compared to solvent exchange of alpha-methylene protons in acyclic, aliphatic sulfonium ions. No other carbon bonded hydrogens in the molecule exchange with solvent under the experimental conditions. Allylic stabilization of a carbanionic-like transition state for the solvent exchange process can account for these results. Solvent exchange under these mild conditions provides a simple way to prepare a 5'-2H-labeled form of the coenzyme analogue.