Allosteric pluripotency as revealed by protein kinase A.

The functional response of a signaling system to an allosteric stimulus often depends on subcellular conditions, a phenomenon known as pluripotent allostery. For example, a single allosteric modulator, Rp-cAMPS, of the prototypical protein kinase A (PKA) switches from antagonist to agonist depending on MgATP levels. However, the mechanism underlying such pluripotent allostery has remained elusive for decades. Using nuclear magnetic resonance spectroscopy, ensemble models, kinase assays, and molecular dynamics simulations, we show that allosteric pluripotency arises from surprisingly divergent responses of highly homologous tandem domains. The differential responses perturb domain-domain interactions and remodel the free-energy landscape of inhibitory excited states sampled by the regulatory subunit of PKA. The resulting activation threshold values are comparable to the effective free energy of regulatory and catalytic subunit binding, which depends on metabolites, substrates, and mutations, explaining pluripotent allostery and warranting a general redefinition of allosteric targets to include specific subcellular environments.

Orientation and effects of mastoparan X on phospholipid bicelles.

Mastoparan X (MPX: INWKGIAAMAKKLL-NH2) belongs to a family of ionophoric peptides found in wasp venom. Upon binding to the membrane, MPX increases the cell's permeability to cations leading to a disruption in the electrolyte balance and cell lysis. This process is thought to occur either through a membrane-thinning mechanism, where the peptide resides on the membrane surface thereby disrupting lipid packing, or through formation of an oligomeric pore. To address this issue, we have used both high-resolution and solid-state 2H NMR techniques to study the structure and orientation of MPX when associated with bicelles. NOESY and chemical shift analysis showed that in bicelles, MPX formed a well-structured amphipathic alpha-helix. In zwitterionic bicelles, the helical axis was found to rest generally perpendicular to the membrane normal, which could be consistent with the "carpet" mechanism for lytic activity. In anionic bicelles, on the other hand, the helical axis was generally parallel to the membrane normal, which is more consistent with the pore model for lytic activity. In addition, MPX caused significant disruption in lipid packing of the negatively charged phospholipids. Taken together, these results show that MPX associates differently with zwitterionic membranes, where it rests parallel to the surface, compared with negatively charged membranes, where it penetrates longitudinally.

Water-macromolecule interactions by NMR: a quadrature-free constant-time approach and its application to CI2.

A pulse sequence is proposed to select water magnetization with enhanced specificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented without quadrature detection, which results in a square root 2 increase in signal-to-noise ratios as compared to traditional non-selective methods for water filtration. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gains in sensitivity. The proposed experiment was applied to unlabeled HEW lysozyme and to 15N-labeled chymotrypsin inhibitor 2 which was partially or non 13C-enriched. Water molecules belonging to a spine of hydration between two pseudo beta-sheet strands were identified, solving previously reported discrepancies between the X-ray and refined NMR structure of CI2. The proposed experiment in particularly suitable for hydration studies of mixtures of labeled and unlabeled components, such as ligand-macromolecule complexes.

Collagen-based structures containing the peptoid residue N-isobutylglycine (Nleu): synthesis and biophysical studies of Gly-Nleu-Pro sequences by circular dichroism and optical rotation.

Single-chain peptide-peptoid structures, Ac-(Gly-Nleu-Pro)n-NH2 (n = 3, 6, and 10) and (Gly-Nleu-Pro)n-NH2 (n = 1 and 9), and template-assembled collagen analogs, KTA-[Gly-(Gly-Nleu-Pro)n-NH2]3 (n = 3 and 6; KTA represents cis,cis-1,3,5-trimethylcyclohexane-1,3, 5-tricarboxylic acid, also known as the Kemp triacid; Nleu denotes N-isobutylglycine), were prepared by solid-phase peptide synthesis methods. Biophysical studies using circular dichroism (CD) and optical rotation measurements show that these collagen analogs form triple-helical conformations when the chain is longer than a critical length. Unlike collagen-based structures composed of Gly-Pro-Hyp and Gly-Pro-Nleu sequences, results reveal that the presence of a positive CD peak between 220 and 225 nm is indicative of triple-helical conformations for these collagen-based structures composed of Gly-Nleu-Pro sequences. Results also indicate that the Gly-Nleu-Pro sequence possesses a higher triple-helical propensity than the Gly-Pro-Nleu sequence as demonstrated by the higher melting temperatures, the faster triple-helix folding, and the lower minimum concentration necessary to detect triple-helicity for the single-chain structures. Therefore, we conclude that the Nleu residue in the second position of the trimeric repeat is more effective in inducing triple-helix formation than Pro in the same position.

Collagen-based structures containing the peptoid residue N-isobutylglycine (Nleu): conformational analysis of Gly-Nleu-Pro sequences by 1H-NMR and molecular modeling.

Molecular modeling and 1H-NMR were employed to study the structure and stability of collagen-like triple helices composed of Gly-Nleu-Pro repeats. The compounds studied include the acetyl analogs Ac-(Gly-Nleu-Pro)n-NH2 (where n = 1, 3, 6, and 10) and the KTA conjugates KTA-[Gly-(Gly-Nleu-Pro)n-NH2]3 (where n = 3 and 6 and KTA denotes the Kemp triacid). The presence of collagen-like assembled structures is supported by a consistent set of experimental observations, which include the appearance of a distinct set of resonances, low hydrogen-exchange rates for Gly NH, cooperative melting transition, and observation of several interchain NOEs. Using 1H-NMR, the triple helicity was monitored as a function of chain length, template, and temperature. These studies show that (Gly-Nleu-Pro)n sequences have a somewhat higher triple-helical propensity than (Gly-Pro-Nleu)n sequences. In addition, our investigations have shown that unlike the triple helices composed of Gly-Pro-Nleu repeats those composed of Gly-Nleu-Pro repeats can access conformations in which the Nleu side chains are arrayed between Pro residues belonging to different triple-helix cross sections. These structural features may serve as a basis for free energy computations and for the study of higher-order structures such as collagen-like fibrils containing peptoid moities.

Lanthionine-somatostatin analogs: synthesis, characterization, biological activity, and enzymatic stability studies.

A series of cyclic somatostatin analogs containing a lanthionine bridge have been subjected to studies of structure-activity relationships. A direct synthesis of the thioether bridged analog (1) of sandostatin (SMS 201,995) and several lanthionine hexa-, hepta-, and octapeptides was carried out by using the method of cyclization on an oxime resin (PCOR) followed by condensation reactions in solution. The structures of the target peptides were analyzed by liquid secondary ion mass spectrometry (LSIMS) and subjected to high-energy collision-induced dissociation (CID) studies after opening of the peptide ring by proteolytic cleavage. The biological activities of these compounds have been evaluated by assaying their inhibitory potencies for the release of growth hormone (GH) from primary cultures of rat anterior pituitary cells, as well as by their binding affinities to cloned somatostatin receptors (SSTR1-5). The structural modification of sandostatin by introducing a lanthionine bridge resulted in a significantly increased receptor binding selectivity. The lanthionine octapeptide with C-terminal Thr-ol (1) showed similar high affinity for rat SSTR5 compared to somatostatin[1-14] and sandostatin. However, it exhibits about 50 times weaker binding affinity for mSSTR2b than sandostatin. Similarly, the lanthionine octapeptide with the C-terminal Thr-NH2 residue (2) has higher affinity for rSSTR5 than for mSSTR2B. Both peptides (compounds 1 and 2) have much lower potencies for inhibition of growth hormone secretion than sandostatin. This is consistent with their affinities to SSTR2, the receptor which is believed to be linked to the inhibition of growth hormone release by somatostatin and its analogs. The metabolic stability of lanthionine-sandostatin and sandostatin have been studied in rat brain homogenates. Although both compounds have a high stability toward enzymatic degradation, the lanthionine analog has a 2.4 times longer half-life than sandostatin. The main metabolites of both compounds have been isolated and identified by using an in vivo technique (cerebral microdialysis) and mass spectrometry.

The solution conformational features of two highly homologous antigenic peptides of foot-and-mouth disease virus serotype A, variant A and USA, correlate with their serological properties.

The solution structure of a peptide corresponding to the VP1 region 141-160 of foot-and-mouth disease virus (FMDV) serotype A variant USA has been studied by NMR and computer calculations and compared with the results from a study on a highly homologous peptide deriving from serotype A, variant A. The two peptides differ in their serological behavior and contain the immunodominant epitope of the virus which partly overlaps with its receptor binding region. Distance constraints, derived both from 2D and 3D homonuclear NMR and 2D-heteronuclear NMR experiments, were combined with DG calculations to yield 50 structures. After refinement through EM and restrained molecular dynamics simulations the selected structures shared several general features. In particular the 151-158 region was a helix in all cases while a large loop similar to that found in peptide A but comprising less residues and stabilized by an H-bond between the side chains of D147 and S150 was found in the majority of structures. A further loop, common to all structures, was identified around the RGD sequence (145-147). This was different from that found in the corresponding region of peptide A as were the conformations of the individual residues within the RGDX sequence. The different structural features shown by the two peptides were rationalized in terms of the S148 (peptide A) to F148 (peptide USA) mutation. The second mutation, that at position 153 (L in A, P in USA) did not appear to affect the structure of the peptide significantly although the different dimensions of the loop in the central region and the type of H-bond stabilizing it could be potentially ascribed to this second mutation. All criteria used pointed to different structural features for the two peptides consistent with their serological behaviour.