ABSTRACT
Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.
ABSTRACT
The T1 domain, a highly conserved cytoplasmic portion at the N-terminus of the voltage-dependent K+ channel (Kv) alpha-subunit, is responsible for driving and regulating the tetramerization of the alpha-subunits. Here we report the identification of a set of mutations in the T1 domain that alter the gating properties of the Kv channel. Two mutants produce a leftward shift in the activation curve and slow the channel closing rate while a third mutation produces a rightward shift in the activation curve and speeds the channel closing rate. We have determined the crystal structures of T1 domains containing these mutations. Both of the leftward shifting mutants produce similar conformational changes in the putative membrane facing surface of the T1 domain. These results suggest that the structure of the T1 domain in this region is tightly coupled to the channel's gating states.
Subject(s)
Aplysia/chemistry , Ion Channel Gating , Potassium Channels/chemistry , Potassium Channels/metabolism , Amino Acid Substitution/genetics , Animals , Conserved Sequence/genetics , Crystallography, X-Ray , Electric Conductivity , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Potassium Channels/genetics , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Shaker Superfamily of Potassium Channels , Structure-Activity Relationship , ThermodynamicsABSTRACT
The N-terminal, cytoplasmic tetramerization domain (T1) of voltage-gated K+ channels encodes molecular determinants for subfamily-specific assembly of alpha-subunits into functional tetrameric channels. Crystal structures of T1 tetramers from Shaw and Shaker subfamilies reveal a common four-layered scaffolding. Within layer 4, on the hypothetical membrane-facing side of the tetramer, the Shaw T1 tetramer contains four zinc ions; each is coordinated by a histidine and two cysteines from one monomer and by one cysteine from an adjacent monomer. The amino acids involved in coordinating the Zn2+ ion occur in a HX5CX20CC sequence motif that is highly conserved among all Shab, Shaw and Shal subfamily members, but is not found in Shaker subfamily members. We demonstrate by coimmunoprecipitation that a few characteristic residues in the subunit interface are crucial for subfamily-specific tetramerization of the T1 domains.