Nano-Scale Alignment of Proteins on a Flexible DNA Backbone

Nano-scale alignment of several proteins with freedom of motion is equivalent to an enormous increase in effective local concentration of proteins and will enable otherwise impossible weak and/or cooperative associations between them or with their ligands. For this purpose, a DNA backbone made of six oligodeoxynucleotide (ODN) chains is designed in which five double-stranded segments are connected by four single-stranded flexible linkers. A desired protein with an introduced cysteine is connected covalently to the 5′-end of azido-ODN by catalyst-free click chemistry. Then, six protein-ODN conjugates are assembled with their complementary nucleotide sequences into a single multi-protein-DNA complex, and six proteins are aligned along the DNA backbone. Flexible alignment of proteins is directly observed by high-speed AFM imaging, and association of proteins with weak interaction is demonstrated by fluorescence resonance energy transfer between aligned proteins.     

Flexible polymer-induced condensation and bundle formation of DNA and F-actin filaments

A simple semi-empirical theory is developed for the ionic strength dependence of the flexible polymer-induced condensation of semiflexible polyelectrolytes such as DNA and F-actin filaments. Critical concentrations of flexible polymer needed for condensation are calculated by comparing the free energies of inserting the semiflexible polyelectrolytes in a solution of flexible polymers, respectively, in their free state, and in their condensed state. Predictions of the theory are compared to experimental data on the condensation of DNA and F-actin filaments induced by the flexible polymer poly(ethylene oxide). The theory also predicts that reentrant decollapse is possible at low ionic strength and high concentrations of flexible polymer, as observed for DNA.     

Flexible 5-guanidino-4-nitroimidazole DNA lesions: structures and thermodynamics

5-Guanidino-4-nitroimidazole (NI), derived from guanine oxidation by reactive oxygen and nitrogen species, contains an unusual flexible ring-opened structure, with nitro and guanidino groups which possess multiple hydrogen bonding capabilities. In vitro primer extension experiments with bacterial and mammalian polymerases show that NI incorporates C as well as A and G opposite the lesion, depending on the polymerase. To elucidate structural and thermodynamic properties of the mutagenic NI lesion, we have investigated the structure of the modified base itself and the NI-containing nucleoside with high-level quantum mechanical calculations and have employed molecular modeling and molecular dynamics simulations in solution for the lesion in B-DNA duplexes, with four partner bases opposite the NI. Our results show that NI adopts a planar structure at the damaged base level. However, in the nucleoside and in DNA duplexes, steric hindrance between the guanidino group and its linked sugar causes NI to be nonplanar. The NI lesion can adopt both syn and anti conformations on the DNA duplex level, with the guanidino group positioned in the DNA major and minor grooves, respectively; the specific preference depends on the partner base. On the basis of hydrogen bonding and stacking interactions, groove dimensions, and bending, we find that the least distorted NI-modified duplex contains partner C, consistent with observed incorporation of C opposite NI. However, hydrogen bonding interactions between NI and partner G or A are also found, which would be compatible with the observed mismatches.     

Actin Filament Length Tunes Elasticity of Flexibly Cross-Linked Actin Networks

Networks of the cytoskeletal biopolymer actin cross-linked by the compliant protein filamin form soft gels that stiffen dramatically under shear stress. We demonstrate that the elasticity of these networks shows a strong dependence on the mean length of the actin polymers, unlike networks with small, rigid cross-links. This behavior is in agreement with a model of rigid filaments connected by multiple flexible linkers.     

A Simple,Flexible Failure Model

A new failure model is introduced in the form of a four-parameter nonlinear differential equation, with failure probability as the dependent variable and failure time as the independent variable. The first parameter characterizes the location, the second the scale, and the other two the shape of the model. The type of the accompanying hazard function is immediately read off the shape parameters. The new model approximates the classical failure models with rather high precision, but also models cases where the failure density is skewed to the left. It can be used to analyze survival data objectively, based on the shape of the failure distribution. The computation of quantiles and moments is easy and fast. Nonlinear regression methods are used to estimate parameter values.     

Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.     

Production Capacity Modeling of Alternative, Nonidentical, Flexible Machines

Analyzing the production capacity of a flexible manufacturing system consisting of a number of alternative, nonidentical, flexible machines, where each machine is capable of producing several different part types simultaneously (by flexibly allocating its production capacity among these part types), is not a trivial task. The production capacity set of such a system is naturally expressed in terms of the machine-specific production rates of all part types. In this paper we also express it in terms of the total production rates of all part types over all machines. More specifically, we express the capacity set as the convex hull of a set of points corresponding to all possible assignments of machines to part types, where in each assignment each machine allocates all its capacity to only one part type. First, we show that within each subset of assignments having a given number of machines assigned to each part type, there is a unique assignment that corresponds to an extreme point of the capacity set. Then, we propose a procedure for generating all the extreme points and facets of the capacity set. Numerical experience shows that when the number of part types is less than four, the size of the capacity set (measured in terms of the number of variables times the number of constraints) is smaller, if the capacity set is expressed in terms of the total production rates of all part types over all machines than if it is expressed in terms of the machine-specific production rates of all part types. When the number of part types is four or more, however, the opposite is true.     

Improved Protocol for the Synthesis of Flexibly Protected Morpholino Monomers from Unprotected Ribonucleosides

An inexpensive and much improved protocol has been developed for the synthesis of protected morpholino monomers from unprotected ribonucleosides in high overall yield, using oxidative glycol cleavage and reductive amination strategy. Unlike the previous methods, the present strategy allows installing the exocyclic amine protections at a later stage, and thus avoids the use of expensive, or commercially unavailable, exocyclic amine-protected ribonucleosides as starting materials. To demonstrate the flexibility of the present method in choosing protecting groups, the monomers have been protected with several such groups of different deblocking properties at the exocyclic amine position.     

Flexible structural comparison allowing hinge-bending, swiveling motions

We present an efficient method for flexible comparison of protein structures, allowing swiveling motions. In all currently available methodologies developed and applied to the comparisons of protein structures, the molecules are considered to be rigid objects. The method described here extends and generalizes current approaches to searches for structural similarity between molecules by viewing proteins as objects consisting of rigid parts connected by rotary joints. During the matching, the rigid subparts are allowed to be rotated with respect to each other around swiveling points in one of the molecules. This technique straightforwardly detects structural motifs having hinge(s) between their domains. Whereas other existing methods detect hinge-bent motifs by initially finding the matching rigid parts and subsequently merging these together, our method automatically detects recurring substructures, allowing full 3 dimensional rotations about their swiveling points. Yet the method is extremely fast, avoiding the time-consuming full conformational space search. Comparison of two protein structures, without a predefinition of the motif, takes only seconds to one minute on a workstation per hinge. Hence, the molecule can be scanned for many potential hinge sites, allowing practically all C(alpha) atoms to be tried as swiveling points. This algorithm provides a highly efficient, fully automated tool. Its complexity is only O(n2), where n is the number of C(alpha) atoms in the compared molecules. As in our previous methodologies, the matching is independent of the order of the amino acids in the polypeptide chain. Here we illustrate the performance of this highly powerful tool on a large number of proteins exhibiting hinge-bending domain movements. Despite the motions, known hinge-bent domains/motifs which have been assembled and classified, are correctly identified. Additional matches are detected as well. This approach has been motivated by a technique for model based recognition of articulated objects originating in computer vision and robotics.     

Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin

Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.     

Epidemiological transitions, reproductive health, and the Flexible Response Model

In concert with improving standards of living since the mid-19th century, chronic and non-infectious diseases replaced infectious diseases as the major causes of mortality in more developed countries. Thus, economic development has been seen as one strategy to improve women's reproductive health. However, rates of two of the major contributors to women's illness, maternal mortality and breast cancer, do not correspond well with the level of economic development. Drawing upon our longitudinal study of reproductive functioning among rural Bolivians (Project Reproduction and Ecology in Provincia Aroma (REPA)), we propose an evolutionary model to explain variation in certain aspects of women's reproductive health. Our findings suggest new avenues of inquiry into the determinants of reproductive health and have implications for improving the well-being of women worldwide.     

Flexible rhombic flap

A flexible rhombic flap which can be designed in accordance with the degree of tension of skin bordering a part of the defect has been described. This procedure provides sufficient coverage of a skin defect with very little dog-ear.