D Risal | PubMed Health

PubMed1999

Multistep denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet.

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi, is a dumbbell-shaped protein in which two globular domains are connected by a three-stranded beta-sheet segment that is solvent-exposed on both faces. Previous studies showed that the whole protein, including the single-layer beta-sheet, is highly rigid. To elucidate the folding mechanism and the role of the central beta-sheet in the formation of the rigid molecule, we investigated the equilibrium thermal denaturation reaction of OspA. We applied differential scanning calorimetry, heteronuclear NMR spectroscopy, and solution small-angle X-ray scattering (SAXS) to characterize the reaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically 15N-labeled at Lys residues identified the locations of the two folding units and revealed that the C-terminal segment is less stable than the remaining N-terminal segment. The boundary between the two folding units is located within the central beta-sheet. The interconversion among the three folding states (fully folded, C-terminus unfolded, and fully denatured) is slow relative to chemical shift differences (<24 Hz), indicating that there are significant kinetic barriers in the denaturation reactions. SAXS measurements determined the radius of gyration of the native protein to be 25.0 +/- 0.3 A, which increases to 34.4 +/- 1.0 A in the first transition, and then to 56.1 +/- 1.6 A in the second transition. Thus, the intermediate state, in which the C-terminal folding unit is already denatured, is still compact. These results provide a basis for elucidating the folding mechanism of OspA.

Antigens, Surface↗

PubMed1999

Dynamic potassium channel distributions during axonal development prevent aberrant firing patterns.

The distribution and function of Shaker-related K+ channels were studied with immunofluorescence and electrophysiology in sciatic nerves of developing rats. At nodes of Ranvier, Na+ channel clustering occurred very early (postnatal days 1-3). Although K+ channels were not yet segregated at most of these sites, they were directly involved in action potential generation, reducing duration, and the refractory period. At approximately 1 week, K+ channel clusters were first seen but were within the nodal gap and in paranodes, and only later (weeks 2-4) were they shifted to juxtaparanodal regions. K+ channel function was most dramatic during this transition period, with block producing repetitive firing in response to single stimuli. As K+ channels were increasingly sequestered in juxtaparanodes, conduction became progressively insensitive to K+ channel block. Over the first 3 weeks, K+ channel clustering was often asymmetric, with channels exclusively in the distal paranode in approximately 40% of cases. A computational model suggested a mechanism for the firing patterns observed, and the results provide a role for K+ channels in the prevention of aberrant excitation as myelination proceeds during development.

Action Potentials↗