Designing sequence to control protein function in an EF-hand protein

The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca(2+)-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca(2+) sensorlike conformational changes. The X-ray crystal structure of Ca(2+)-CBM-1 determined at 1.44 A resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.     

Synthesis and Photophysics of Core‐Substituted Naphthalene Diimides: Fluorophores for Single Molecule Applications

The synthesis and photophysics of two new aminopropenyl naphthalene diimide (SANDI) dyes are reported. A general and convenient method for the synthesis of the precursor mono‐, di‐, and tetrabrominated 1,4,5,8‐naphthalene tetracarboxylic dianhydrides is described. The two core‐substituted SANDIs exhibit many of the photophysical properties required for fluorescence labeling applications including high photostability and high fluorescence quantum yields (>0.5) in the visible region of the spectrum. The emission wavelength is sensitive to the number of substituents on the NDI core, and the fluorescence decay times are in the range of ～8–12 ns for both compounds in the solvents investigated. Preliminary fluorescence emission data from single molecules of the compounds embedded in poly(methyl methacrylate) films are also reported and show that single molecules have very low yields of photobleaching, particularly the di‐substituted system. Furthermore, only a small proportion (<10 %) of the single molecules studied display fluorescence intermittencies or “blinks” in their photon trajectory. The compounds appear to be excellent candidates for applications at the single molecule level, for example, as FRET labels.     

Uniform Uniform Exponential Growth of Subgroups of the Mapping Class Group

Let Mod(S) denote the mapping class group of a compact, orientable surface S. We prove that finitely generated subgroups of Mod(S) which are not virtually abelian have uniform exponential growth with minimal growth rate bounded below by a constant depending only, and necessarily, on S. For the proof, we find in any such subgroup explicit free group generators which are “short” in any word metric. Besides bounding growth, this allows a bound on the return probability of simple random walks.