Eric S. Peterson

Probing the Conformation of Hemoglobin Presbyterian in the R-State

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108(β)->Lys mutation–induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the α1β1 and α1β2 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(α) of Tg-HbP, a residue of the α1β1 interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the α1β2 interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(β). In short, Lys-108(β) in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbAs affinity to oxygen.

Frequency-dependent coupling between rhythmically active neurons in the leech

The heart excitor (HE) cells, a set of rhythmically active motor neurons, drive the heartbeat of the medicinal leech. Their activity is gated by inhibitory input from a network of interneurons, but that influence may be modified locally by electrotonic coupling between the HE cells. In this paper I analyze that electrotonic coupling by applying direct current and alternating current signals, and compare the results with predictions based on linear cable theory. The electrotonic junction itself appears to be conventional, but because of the membrane properties of the HE cells, the coupling strength depends upon both the frequency and polarity of the signal and the phase of heartbeat cycle when the signal is applied.

The Effect of Hofmeister Ions on the Folding Pathway of Cytochrome C during Thermal or Chemical Denaturization

The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of ions from the Hofmeister series is examined. Unfolding was initiated both thermally and with chemical denaturants. Hofmeister ions were added singly and in pairs to alter the stability of the native folded state, the unfolded state, and two partially folded intermediates. Protein stability was characterized by either the midpoint of the chemical denaturization curve or by the melting temperature in the thermal studies. UV/VIS absorption spectroscopy and a basis spectra fitting analysis were used to determine the populations of each protein conformation along the folding pathway. These species can be differentiated by their axial heme ligands. Four species exist in solution: the native HM state (His18/Met80), the partially folded HW (His18/water) and HH (His18/His33) intermediates, and the 5C (water) unfolded state. The results indicate that the thermal and chemical denaturization pathways are not the same and that both involve significant backbone rearrangement. The relative populations of the conformational states depends on how the protein is denatured. Additionally, it was found that addition of multiple ions changed the proteins stability in an additive manner. These results are discussed in terms of the hydrophobic effect, partitioning of the ions to the protein surface, and an altered water structure around the protein.

The Effects of Hofmeister Salts on the Cytochrome c Folding Pathway in Solution and within Sol-Gel Glasses

The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of steric constraints and altered solvating water structure is examined. Sol-gel encapsulation was used to constrain the protein to a volume slightly larger than the native state. Hofmeister salts were added to alter water order. UV/VIS absorption spectroscopy and a basis spectra fitting analysis were used to determine the populations of species present along the folding pathway. These species can be differentiated by their axial heme ligands. Four species exist in solution: the native HM state (His18/Met80), the partially folded HW (His18/water) and HH (His18/His33) intermediates, and the 5C (water) unfolded state. An additional unfolded state found only within the sol-gel contains an unligated four-coordinate heme sequestered from aqueous solution. In solution, the native HM state unfolds primarily into the HH state, while unfolding within the gel produces comparable amounts of the HH, HW, and four-coordinate states. This indicates that the steric constraints within the gel pores hinder some backbone motions. Four anions (HPO4−2, H2PO4−1, SO4−2 and Cl−1) and three cations (Gdn+1, K+1, and NH4+1) that lie within the Hofmeister series were utilized. Gdn+1 lies at one end of the series and promotes unfolding of the protein. Presence of an additional ion can counter the denaturing effects of the Gdn+1 to an extent dependant on its proximity to Gdn+1 in the series. Both the (un)folding kinetics and the accessible conformations were found to depend on the identity of ions present. A model discussing changes in protein stability as a function of water order and the hydrophobic effect is used to interpret these results. Water order depends on the degree of confinement in the gel pores and the properties of the ions present.