No abstract

Proline is established as a potent breaker of both et-helical and 13-sheet structures in soluble (globular) proteins.

Ion-coupled membrane-transport proteins, or secondary transporters, comprise a diverse and abundant group of membrane proteins that are found in all organisms. These proteins facilitate solute accumulation and toxin removal against concentration gradients using energy supplied by ion gradients across membranes. NhaA is a Na+/H+ antiporter of relative molecular mass 42,000, which is found in the inner membrane of Escherichia coli, and which has been cloned and characterized. NhaA uses the H+ electrochemical gradient to expel Na+ from the cytoplasm, and functions primarily in the adaptation to high salinity at alkaline pH. Most secondary transporters, including NhaA, are predicted to have 12 transmembrane helices. Here we report the structure of NhaA, at 7 A resolution in the membrane plane and at 14 A vertical resolution, determined from two-dimensional crystals using electron cryo-microscopy. The three-dimensional map of NhaA reveals 12 tilted, bilayer-spanning helices. A roughly linear arrangement of six helices is adjacent to a compact bundle of six helices, with the density for one helix in the bundle not continuous through the membrane. The molecular organization of NhaA represents a new membrane-protein structural motif and offers the first insights into the architecture of an ion-coupled transport protein.

^Roline is the least likely of the 20 amino acids to be present in an -helix on the basis of analysis of soluble proteins (Chou & Fasman, 1978; O'Neil & DeGrado, 1990), and since it is generally located in hydrophilic environments (Richardson & Richardson, 1989), it would not be expected to occur within a membrane. Nonetheless, Pro has been found to occur with relatively high frequency in the putatively -helical transmembrane (TM) segments of many integral membrane proteins that function as receptor subunits or transporters (Brandi & Deber, 1986). While the distribution of Pro in membranes appears to be random with respect to position within TM segments (Brandi & Deber, 1986), TM Pro residues tend to be conserved among homologous proteins [e.g., Vilsen et al., (1989)].We examine the potential roles of Pro residues in TM helices and assemble this material into a framework upon which the function(s) of individual membrane-buried Pro residues can be assessed. In the ensuing discussion, we categorize the effect(s) of Pro in a TM helix as structural and/or dynamic.Structural roles include purely static effects such as that arising from a kinked helix, as well as electronic effects that stem from increased local polarity. Dynamic roles, which describe the participation of Pro in conformational change, are in two categories: cis-trans isomerization of Xaa-Pro peptide bonds and Pro-mediated interconversions among all-trans conformational states. Structural Effects of Proline Residues in TransmembraneHelices. Proline is the only mammalian imino acid. As such, its side chain is bonded to the tertiary nitrogen in a cyclic pyrrolidine ring. This effectively fixes the backbone dihedral phi ( ) angle at the preferred -helical value, -60°, and leaves the psi ( ) angle flexible [with two preferred (so-called cis'/trans') regions] but hindered. As well, the bulky ring tThis work was supported, in part, by grants to C.M.D. from the Medical Research Council of Canada (MRC MT-5810) and the Natural Sciences and Engineering Research Council of Canada (NSERC A2807). K.A.W. holds an NSERC studentship.

Electron cryomicroscopy of frozen-hydrated twodimensional crystals of NhaA, a Na ⍣ /H ⍣ antiporter from Escherichia coli predicted to have 12 transmembrane α-helices, has facilitated the calculation of a projection map of NhaA at 4.0 Å resolution. NhaA was homologously expressed in E.coli with a His 6 tag, solubilized in dodecyl maltoside and purified in a single step using Ni 2⍣ affinity chromatography. Twodimensional crystals were obtained after reconstitution of purified protein with E.coli lipids. The projection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of~38⍥48 Å with a ringshaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices. Two crystal forms with p22 1 2 1 symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers. This work provides the first direct glimpse into the structure of a secondary transporter.

The structure and dynamics of the 53-residue filamentous bacteriophage IKe major coat protein in fully protonated myristoyllysophosphatidylglycerol (MPG) micelles were characterized using multinuclear solution NMR spectroscopy. Detergent-solubilized coat protein [sequence: see text] mimics the membrane-bound "assembly intermediate" form of the coat protein which occurs during part of the phage life cycle. NMR studies of the IKe coat protein show that the coat protein is largely alpha-helical, exhibiting a long amphipathic surface. helix (Asn 4 to Ser 26) and a shorter "micelle-spanning" C-terminal helix which begins at TRP 29 and continues at least to Phe 48. Pro 30 likely occurs in the first turn of the C-terminal helix, where it is ideally situated given the hydrogen bonding and steric restrictions imposed by this residue. The similarity of 15N relaxation values (T1, T2, and NOE and 500 MHz and T2 at 600 MHz) among much of the N-terminal helix and all of the TM helix indicates that the N-terminal helix is as closely associated with the micelle as the TM helix. The description of the protein in the micelle is supported by the observation of NOEs between lysolipid protons and protein amide protons between asn 8 and Ser 50. The N-terminal and TM helices exhibit substantial mobility on the microsecond to second time scale, which likely reflects changes in the orientation between the two helices. The overall findings serve to clarify the role of individual residues in the context of a TM alpha-helix and provide an understanding of the secondary structure, dynamics, and aqueous and micellar environments of the coat protein.

The contributions of crop wild relatives (CWR) to food security depend on their conservation and accessibility for use. The United States contains a diverse native flora of CWR, including those of important cereal, fruit, nut, oil, pulse, root and tuber, and vegetable crops, which may be threatened in their natural habitats and underrepresented in plant conservation repositories. To determine conservation priorities for these plants, we developed a national inventory, compiled occurrence information, modeled potential distributions, and conducted threat assessments and conservation gap analyses for 600 native taxa. We found that 7.1% of the taxa may be critically endangered in their natural habitats, 50% may be endangered, and 28% may be vulnerable. We categorized 58.8% of the taxa as of urgent priority for further action, 37% as high priority, and 4.2% as medium priority. Major ex situ conservation gaps were identified for 93.3% of the wild relatives (categorized as urgent or high priority), with 83 taxa absent from conservation repositories, while 93.1% of the plants were equivalently prioritized for further habitat protection. Various taxonomic richness hotspots across the US represent focal regions for further conservation action. Related needs include facilitating greater access to and characterization of these cultural-genetic-natural resources and raising public awareness of their existence, value, and plight.

Adults and infants were tested for the capacity to detect correspondences between nonspeech sounds and real vowels. The /i/ and /a/ vowels were presented in 3 different ways: auditory speech, silent visual faces articulating the vowels, or mentally imagined vowels. The nonspeech sounds were either pure tones or 3-tone complexes that isolated a single feature of the vowel without allowing the vowel to be identified. Adults perceived an orderly relation between the nonspeech sounds and vowels. They matched high-pitched nonspeech sounds to /i/ vowels and low-pitched nonspeech sounds to /a/ vowels. In contrast, infants could not match nonspeech sounds to the visually presented vowels. Infants' detection of correspondence between auditory and visual speech appears to require the whole speech signal; with development, an isolated feature of the vowel is sufficient for detection of the cross-modal correspondence.