63ª Reunião Anual da SBPC

C. Ciências Biológicas - 1. Biofísica - 3. Biofísica Molecular

C2A DOMAIN OF SYNAPTOTAGMIN 1: MEMBRANE BINDING STUDIES BY SDSL

Sheila Gonçalves do Couto 1 Dawn Z. Herrick 2 David S. Cafiso 2

1. Prof.(a) Dr.(a) Instituto de Física, Universidade Federal de Goiás - UFG 2. Prof.(a) Dr.(a) Department of Chemistry and Biophysics, University of Virginia - UVA

INTRODUÇÃO:

Synaptotagmin 1 contains two C2 domains, C2A and C2B, connected by a flexible linker. These domains bind Ca2+, and once Ca2+-bound, are able to penetrate negatively-charged membranes. Based on a few early NMR studies on syt1 and other C2 domain-containing proteins, it was hypothesized that the structure of the C2 domain does not change upon membrane-binding. However, EPR line shape analysis shows that when the C2 domains are membrane-bound, there is broadening in β-strand sites. From depth data (power saturation), these same sites appear to not be involved in membrane-contact, but are aqueous exposed. One explanation of this phenomenon, is that the C2 domain’s β-strands undergo a structural re-arrangement upon membrane binding. In this report, we have investigated this idea by measuring distances between double mutants in the C2A domain using EPR when the protein is in solution (with Ca2+) and membrane-bound (with Ca2+).

METODOLOGIA:

Lyophilized protein was available for three of these double mutants, G175CL202C, K189CF210C, and F193CF210C. Spin labeling and subsequent purification was carried out as done regularly for C2A and 3:1 POPC:POPS LUVs were made. Modeling of the interspin distances between the nitrogen of the appended nitroxides of MTSL was done in InsightII. First, MTSL was appended to each of the five sites investigated: K189C, F210C, F193C, G175C, and L202C. Next, the dihedral angles of MTSL were changed to those rotamers found to be most populated based on crystallography studies of spin-labeled proteins.

RESULTADOS:

G175R1L202R1, showed the greatest dipolar broadening. The distance determined by cw-EPR, 12 Å, closely agreed with the modeled distance. The line shape was fit well by modeling the dipolar broadening using Short Distances (SD), and due to a lack of protein. To the double mutant, K189R1F210R1, the line shape of the double labeled protein, does indicate some dipolar broadening, and distances derived by cw-EPR and DEER agree well and are in the range of 13-16 Å. The dipolar evolution of K189R1F210R1 has some interesting features: a sharp decay and prominent periodic oscillation that is most likely due to ESEEM. To the double mutant, F193R1F210R1, SD yielded a Gaussian of 14 Å. The best fit for R1R1’ was a distribution at 15-16 Å. Indeed the line shape of the double mutant F193R1F210R1 contains a sharp component, noticeable at both the low and high field lines. The DEER data for F193R1F210R1 makes the situation even more perplexing. For the protein in solution, the mean distance for fitting to one Gaussian is 31 Å. This does not change depending on the background subtraction and also when the delay time was extended from 1.2 μs to 2.0 μs. Obviously, this does not agree with the cw-EPR determined distance nor the estimated distance derived from modeling the position of R1.

CONCLUSÃO:

From investigating these three C2A double mutants, G175L202, K189F210, and F193F210, it appears that the distance between the two sites does not change when the protein is in solution with Ca2+ to being membrane-bound with Ca2+. This agrees with early NMR work.

Palavras-chave: Synaptotagmin, C2A Domain, SDSL.