Electric birefringence of recombinant spectrin segments 14, 14-15, 14-16, and 14-17 from Drosophila alpha-spectrin

Members of the spectrin protein family can be found in many different cells and organisms. In all cases studied, the major functional role of these pr oteins is believed to be structural rather than enzymatic. All spectrin pro teins are highly elongated and consist mainly of homologous repeats that co nstitute rigid segments connected in tandem. It is commonly believed that t he details of the spectrin function depend critically on the flexibility of the links between the segments. Here we report on a work addressing this q uestion by studying the transient electric birefringence of recombinant spe ctrin fragments consisting of segments 14, 14-15, 14-16, and 14-17, respect ively, from Drosophila alpha-spectrin. Transient electric birefringence dep ends sharply on both molecular length and flexibility. We found that the bi refringence relaxation time of segment 14 measured at 4 degrees C, but scal ed to what is expected at 20 degrees C, equals 16 ns (+/-15%) at pH 7.5 and ionic strength 6 mM. This is consistent with this single segment being rig id, 5 nm long and having an axial ratio equal to about two. Under the same conditions, segments 14-15, 14-16 and 14-17 show relaxation times of 45, 39 and 164 ns (all +/- 20%), respectively, scaled to what is expected at 20 d egrees C. When the temperature is increased to 37 degrees C the main relaxa tion time for each of these multisegment fragments, scaled to what is expec ted at 20 degrees C, increased to 46, 80, and 229 ns (all +/- 20%), respect ively. When the ionic strength and the Debye shielding is low, the dynamics of these short fragments even at physiological temperature is nearly the s ame as for fully extended weakly bending rods with the same lengths and axi al ratios. When the ionic strength is increased to 85 mM, the main relaxati on time for each of these multisegment fragments is reduced 20-50% which su ggests that at physiological salt and temperature conditions the links in 2 -4-segment-long fragments exhibit significant thermally induced flexing. Pr ovided that the recombinant spectrin fragments can serve as a model for nat ive spectrin, this implies that, at physiological conditions, the overall c onformational dynamics of a native spectrin protein containing 20-40 segmen ts equals that of a flexible polymer. (C) 1999 Elsevier Science B.V. All ri ghts reserved.