Sebastian Doniach

Use of one‐electron theory for the interpretation of near edge structure in K‐shell x‐ray absorption spectra of transition metal complexes

We report the results of multiple scattered wave SCF X‐alpha calculations of the one‐electron cross section for K‐shell photoabsorption in the molecular complexes MoO4−−, CrO4−−, and MoS4−−. We show that the method can successfully account for energy separations and relative cross sections of spectral features both below and above the K‐shell ionization threshold. Furthermore, we show: (a) that the first fairly intense peak on the low energy side of the rising edge for molybdate and chromate is due to a dipole allowed transition to a bound antibonding state of mainly nd character on the metal ion; this transition is possible because of the mixing with the ligand p orbitals having the proper T2 symmetry induced by the tetrahedral molecular potential; (b) the shoulder on the rising absorption edge can be explained by the beginning of the steplike continuum absorption when convolved with a Lorentzian function of frequency to imitate lifetime and monochromator broadening: (c) the main absorption peak is due t...

Exploring the folding landscape of a structured RNA

Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

Rapid compaction during RNA folding

We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

A comparative study of motor-protein motions by using a simple elastic-network model

In this work, we report on a study of the structure-function relationships for three families of motor proteins, including kinesins, myosins, and F1-ATPases, by using a version of the simple elastic-network model of large-scale protein motions originally proposed by Tirion [Tirion, M. (1996) Phys. Rev. Lett. 77, 1905–1908]. We find a surprising dichotomy between kinesins and the other motor proteins (myosins and F1-ATPase). For the latter, there exist one or two dominant lowest-frequency modes (one for myosin, two for F1-ATPase) obtained from normal-mode analysis of the elastic-network model, which overlap remarkably well with the measured conformational changes derived from pairs of solved crystal structures in different states. Furthermore, we find that the computed global conformational changes induced by the measured deformation of the nucleotide-binding pocket also overlap well with the measured conformational changes, which is consistent with the “nucleotide-binding-induced power-stroke” scenario. In contrast, for kinesins, this simplicity breaks down. Multiple modes are needed to generate the measured conformational changes, and the computed displacements induced by deforming the nucleotide-binding pocket also overlap poorly with the measured conformational changes, and are insufficient to explain the large-scale motion of the relay helix and the linker region. This finding may suggest the presence of two different mechanisms for myosins and kinesins, despite their strong evolutionary ties and structural similarities.

Observation of an electric quadrupole transition in the X-ray absorption spectrum of a Cu(II) complex

Abstract The polarized X-ray absorption cross section of the ls → 3d transition in a square planar CuCl2−4 complex has been measured with respect to rotation about an axis normal to the CuCl4. The cross section exhibits four-fold periodicity about this axis, indicating that the transition is primarily due to coupling with the electric quadrupole component of the radiation. The vibronically allowed dipole transition is approximately one-third as the quadrupolar cross section. These observations are in agreement with SCF Xα multiple-scattered wave calculations, and may have implications for the intepretation of other X-ray absorption spectra. The half-filled d orbital is shown to have the angular characteristics of dx2−y2.

Toward a taxonomy of the denatured state: small angle scattering studies of unfolded proteins.

Publisher Summary This chapter reviews recent small-angle X-ray and neutron scattering (SAXS and SANS) studies of putatively “fully” unfolded states formed at equilibrium. It discusses the taxonomy of unfolded states that is chemically denatured state, thermally denatured state, pressure-denatured state, cold-unfolded states, and intrinsically unfolded proteins. The majority of SAXS and SANS studies of the unfolded state focus on the ensembles of states induced by chemical denaturants such as urea, GuHCl, extremes of pH, and organic cosolvents. As urea and GuHCl dominate spectroscopic studies of protein folding thermodynamics and kinetics, these denaturants have similarly been employed in the vast majority of small-angle scattering studies as well. The extraordinary solubility of unfolded proteins at high levels of urea or GuHCl provides an added technical benefit. Limited SAXS studies suggest that urea and GuHCl produce indistinguishable denatured states. Studies of thermally denatured proteins remain technically challenging owing to the propensity of thermally unfolded proteins to aggregate. Despite this potential difficulty, small-angle scattering techniques have been employed in the characterization of a number of thermally unfolded states. Because the molar volume of an unfolded protein is less than that of the native state, increasing pressure leads to denaturation. High-pressure SAXS was employed to monitor the pressure-induced unfolding of Snase.

Thermodynamic fluctuations in phospholipid bilayers

A simplified statistical mechanical model of the solid–fluid melting transition of wet lipid bilayers is developed which allows for calculation of thermodynamic fluctuation effects. The model is used to estimate the lowering of the free energy barrier for transbilayer ion permeability due to enhanced lateral compressibility near the melting point. The calculation is used to fit permeability data for dipalmitoyl phosphatidyl choline vesicles and the fit provides evidence that the bilayer is quite close to a critical temperature at which the melting transition would go from first to second order.

Reconstruction of low-resolution three-dimensional density maps from one-dimensional small-angle X-ray solution scattering data for biomolecules

A Monte Carlo type reconstruction algorithm (Saxs3D) to yield low-resolution three-dimensional structures from one-dimensional small-angle X-ray scattering data (SAXS) is presented. It is demonstrated that Saxs3D reliably reproduces the shape of several test protein structures, with their respective SAXS profiles calculated theoretically from their known high-resolution atomic coordinate sets. A reconstruction for experimentally obtained scattering data for GroEL, a molecular chaperone, correctly reproduced the gross structural features of GroEL. Compared to other reconstruction methods described in the literature, Saxs3D has the advantage of allowing for any topology of the target structure, does not require any prior estimation of its dimensions, is fast and conceptually very simple.

Small angle X-ray scattering reveals a compact intermediate in RNA folding

We have used small angle X-ray scattering (SAXS) to monitor changes in the overall size and shape of the Tetrahymena ribozyme as it folds. The native ribozyme, formed in the presence of Mg2+, is much more compact and globular than the ensemble of unfolded conformations. Time-resolved measurements show that most of the compaction occurs at least 20-fold faster than the overall folding to the native state, suggesting that a compact intermediate or family of intermediates is formed early and then rearranges in the slow steps that limit the overall folding rate. These results lead to a kinetic folding model in which an initial ‘electrostatic collapse’ of the RNA is followed by slower rearrangements of elements that are initially mispositioned.

Long single α-helical tail domains bridge the gap between structure and function of myosin VI

Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take ∼36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single α-helix of ∼10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the ∼36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.