Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20-presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR 15N relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.

To determine the factors affecting the ground-state electron configuration of low-spin Fe(III) porphyrin complexes, we have examined the (1)H NMR, (13)C NMR, and EPR spectra of a series of low-spin bis-ligated Fe(III) porphyrin complexes [Fe(Por)L(2)](+/-), in which the positions of porphyrin substituents and the coordination ability of axial ligands are different. The seven porphyrins used in this study are meso-tetraalkylporphyrins (TRP: R is propyl, cyclopropyl, or isopropyl), meso-tetraphenylporphyrin (TPP), meso-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin, and 5,10,15,20-tetraphenyl-2,3,7,8,12,13,17,18-octaalkylporphyrins (ORTPP: R is methyl or ethyl). The porphyrin cores of TRP are more or less S(4)-ruffled depending on the bulkiness of the alkyl substituents, while those of ORTPP are highly S(4)-saddled. Three types of axial ligands are examined which have the following characteristics in ligand field theory: they are (i) strong sigma-donating imidazole (HIm), (ii) strong sigma-donating and weak pi-accepting cyanide (CN(-)), and (iii) weak sigma-donating and strong pi-accepting tert-butyl isocyanide ((t)BuNC). In the case of the bis(HIm) complexes, only the isopropyl complex, [Fe(T(i)PrP)(HIm)(2)](+), has shown the less common (d(xz), d(yz))(4)(d(xy))(1) ground state; the other six complexes have exhibited the common (d(xy))(2)(d(xz), d(yz))(3) ground state. When the axial imidazole is replaced by cyanide, even the propyl and cyclopropyl complexes have shown the (d(xz), d(yz))(4)(d(xy))(1) ground state; the TPP and ORTPP complexes have still maintained the common (d(xy))(2)(d(xz), d(yz))(3) ground state. In the case of the bis((t)()BuNC) complexes, all the complexes have shown the (d(xz), d(yz))(4)(d(xy))(1) ground state. However, the contribution of the (d(xz), d(yz))(4)(d(xy))(1) state to the electronic ground state differs from complex to complex; the (d(xz), d(yz))(4)(d(xy))(1) contribution is the largest in [Fe(T(i)PrP)((t)()BuNC)(2)](+) and the smallest in [Fe(OETPPP)((t)BuNC)(2)](+). We have then examined the electronic ground state of low-spin [Fe(OEP)((t)BuNC)(2)](+) and [Fe(ProtoIXMe(2))((t)BuNC)(2)](+); OEP and ProtoIXMe(2) represent 2,3,7,8,12,13,17,18-octaethylporphyrin and protoporphyrin-IX dimethyl ester, respectively. These porphyrins have a(1u) HOMO in contrast to the other seven porphyrins that have a(2u) HOMO. The (13)C NMR and EPR studies have revealed that the contribution of the (d(xz), d(yz))(4)(d(xy))(1) state in these complexes is as small as that in [Fe(OETPP)((t)BuNC)(2)](+). On the basis of these results, we have concluded that the low-spin iron(III) porphyrins that have (i) strong axial ligands, (ii) highly saddle shaped porphyrin rings, (iii) porphyrins with a(1u) HOMO, and (iv) electron withdrawing substituents at the meso positions tend to maintain the common (d(xy))(2)(d(xz), d(yz))(3) ground state.

Voles and lemmings show extensive variation in population dynamics regulated across and within species. In an attempt to develop and test generic hypotheses explaining these differences, we studied 84 populations of the gray-sided vole (Clethrionomys rufocanus) in Hokkaido, Japan. We show that these populations are limited by a combination of density-independent factors (such as climate) and density-dependent processes (such as specialist predators). We show that density-dependent regulation primarily occurs in winter months, so that populations experiencing longer winters tend to have a stronger delayed density-dependence and, as a result, exhibit regular density cycles. Altogether, we demonstrate that seasonality plays a key role in determining whether a vole population is cyclic or not.Clethrionomys rufocanus ͉ seasonal and annual density dependence ͉ state-space modeling ͉ sampling variance A long controversy over the issue of density-dependent versus -independent population regulation has led to the conclusion that both factors are important for understanding population fluctuations (1-8). It is, however, less clear how such density-dependent and -independent factors interact with each other in shaping the dynamic pattern of populations across a larger part of the species range. In an attempt to disentangle these issues, we analyze a set of 30-year seasonal (spring and fall) time series from 84 populations of the gray-sided vole [Clethrionomys rufocanus (Sundevall, 1846)] from Hokkaido, Japan (Fig. 1A) (9, 10). To investigate the role of seasonality in the generation of population cycles, we decompose the annual (fall-to-fall) density dependence, as well as the densityindependent stochasticity into their seasonal components. The added detail provided by pinpointing the seasonal arena of population regulation (see supporting information on the PNAS web site, www.pnas.org) provides us with a better basis for suggesting and evaluating hypotheses about the biological mechanisms that cause density dependence, stochasticity, and population f luctuations.A perennial problem in the study of population dynamics has been the relative lack of extensive and accurate data. In this study we attempt to reach more accurate conclusions than are usually possible by two means. First, we use comparative time-series data from a large number of very similar populations. Second, we address the very substantial problem of biased and imprecise measures of population density through use of a state-space modeling approach (11-13), where time-series observations are related to unobserved ''states'' of the real population through a probabilistic observation model accounting for sampling variation. Our study confirms and extends an earlier study of ours (14); whereas the earlier study used only fall data (and as a result could cover the entire island of Hokkaido), the present study used both fall and spring data. The greater detail of the data used in this paper makes a much more detailed analysis of the seasonal structure possible; a p...

C NMR, and EPR studies of a series of low-spin (meso-tetraalkylporphyrinato)iron(III) complexes, [Fe(TRP)(L) 2 ]X where R ) n Pr, c Pr, and i Pr and L represents axial ligands such as imidazoles, pyridines, and cyanide, have revealed that the ground-state electron configuration of [Fe(T n PrP)(L) 2 ]X and [Fe(T c PrP)(L) 2 ]X is presented either as the common (d xy ) 2 (d xz ,d yz ) 3 or as the less common (d xz ,d yz ) 4 (d xy ) 1 depending on the axial ligands. The ground-state electron configuration of the isopropyl complexes [Fe(Ti-PrP)(L) 2 ]X is, however, presented as (d xz ,d yz ) 4 (d xy ) 1 regardless of the kind of axial ligands. In every case, the contribution of the (d xz ,d yz ) 4 (d xy ) 1 state to the electronic ground state increases in the following order: HIm < 4-Me 2 NPy < 2-MeIm < CN -< 3-MePy < Py < 4-CNPy. Combined analysis of the 13 C and 1 H NMR isotropic shifts together with the EPR g values have yielded the spin densities at the porphyrin carbon and nitrogen atoms. Estimated spin densities in [Fe(T i PrP)(4-CNPy) 2 ] + , which has the purest (d xz ,d yz ) 4 (d xy ) 1 ground state among the complexes examined in this study, are as follows: meso-carbon, +0.045; R-pyrrole carbon, +0.0088; β-pyrrole carbon, -0.00026; and pyrrole nitrogen, +0.057. Thus, the relatively large spin densities are on the pyrrole nitrogen and meso-carbon atoms. The result is in sharp contrast to the spin distribution in the (d xy ) 2 (d xz ,d yz ) 3 type complexes; the largest spin density is at the β-pyrrole carbon atoms in bis(1methylimidazole)(meso-tetraphenylporphyrinato)iron(III), [Fe(TPP)(1-MeIm) 2 ] + , as determined by Goff. The large downfield shift of the meso-carbon signal, δ +917.5 ppm at -50 °C in [Fe(T i PrP)(4-CNPy) 2 ] + , is ascribed to the large spin densities at these carbon atoms. In contrast, the large upfield shift of the R-pyrrole carbon signal, δ -293.5 ppm at the same temperature, is caused by the spin polarization from the adjacent mesocarbon and pyrrole nitrogen atoms.