Temperature‐induced unfolding behavior of proteins studied by tensorial elastic network model

Motivated by single molecule experiments and recent molecular dynamics (MD) studies, we propose a simple and computationally efficient method based on a tensorial elastic network model to investigate the unfolding pathways of proteins under temperature variation. The tensorial elastic network model, which relies on the native state topology of a protein, combines the anisotropic network model, the bond bending elasticity, and the backbone twist elasticity to successfully predicts both the isotropic and anisotropic fluctuations in a manner similar to the Gaussian network model and anisotropic network model. The unfolding process is modeled by breaking the native contacts between residues one by one, and by assuming a threshold value for strain fluctuation. Using this method, we simulated the unfolding processes of four well‐characterized proteins: chymotrypsin inhibitor, barnase, ubiquitein, and adenalyate kinase. We found that this step‐wise process leads to two or more cooperative, first‐order‐like transitions between partial denaturation states. The sequence of unfolding events obtained using this method is consistent with experimental and MD studies. The results also imply that the native topology of proteins “encrypts” information regarding their unfolding process. Proteins 2016; 84:1767–1775. © 2016 Wiley Periodicals, Inc.     

Stacking interactions in RNA and DNA: Roll‐slide energy hyperspace for ten unique dinucleotide steps

Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT‐D by ωB97X‐D/6‐31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B‐DNA, whereas, smaller twist values correspond to higher stability to RNA and A‐DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B‐DNA or A‐DNA conformations. Conformational preference of BII sub‐state in B‐DNA is preferentially displayed mainly by pyrimidine–purine steps and partly by purine–purine steps. The purine–pyrimidine steps show largest effect of 5‐methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 134–147, 2015.     

Terminal Twist-Induced Writhe of DNA with Intrinsic Curvature

Supercoiling of a closed circular DNA rod may result from an application of terminal twist to the DNA rod by cutting the rod, rotating one of the cut faces as the other being fixed and then sealing the cut. According to White's formula, DNA supercoiling is probably accompanied by a writhe of the DNA axis. Deduced from the elastic rod model for DNA structure, an intrinsically straight closed circular DNA rod does not writhe as subject to a terminal twist, until the number of rotation exceeds a rod-dependent threshold. By contrast, a closed circular DNA rod with intrinsic curvature writhes instantly as subject to a terminal twist. This noteworthy character in fact belongs to many intrinsically curved DNA rods. By solving the dynamic equations, the linearization of the Euler–Lagrange equations governing intrinsically curved DNA rods, this paper shows that almost every clamped-end intrinsically curved DNA rod writhes instantly when subject to a terminal twist (clamped-end DNA rods include closed circular DNA rods and topological domains of open DNA rods). In terms of physical quantities, the exceptions are identified with points in ℝ⁶ whose projections onto ℝ⁵ (through ignoring the total energy density of a rod) form a subset of a quadratic hypersurface. This paper also suggests that the terminal twist induced writhe is due to the elasticity and the clamped-end boundary conditions of the DNA rods. To my sister for her 50th birthday.     

Nucleosomes in solution exist as a mixture of twist-defect states

The 2.0 A crystal structure of a nucleosome core particle in complex with a bivalent pyrrole-imidazole polyamide reveals that this "clamp" effectively crossbraces the two gyres of the DNA superhelix, thereby stabilizing the nucleosome against dissociation. Using X-ray crystallography and footprinting techniques, we show that the clamp preferentially binds nucleosomes over free DNA, and that nucleosomal DNA exists as a mixture of multiple twist-defect intermediates in solution. The nucleosomes exist in one of two different conformations in various crystal structures that trap twist-defect intermediates, even on a strong positioning sequence. Evidence has been obtained supporting the existence of twist-defect states in nucleosomal DNA in solution that are similar to those obtained in crystal structures. Our results also substantiate the idea that twist diffusion may represent an important means of altering the accessibility of nucleosomal DNA both in the presence and in the absence of ATP-dependent chromatin-remodelling enzymes.     

Effects of small neutral osmolytes on the supercoiling free energy and intrinsic twist of p30delta DNA

Both theory and experiments are employed to investigate the effects of small neutral osmolytes on the average intrinsic twist (l0), the torsion and bending elastic constants, and the twist energy parameter (ET) that governs the supercoiling free energy. The experimental data for ethylene glycol and acetamide at 37 degrees C suggest, and are interpreted in terms of, a model wherein the DNA exhibits an equilibrium between two distinct conformational states that possess different numbers of bound water molecules and exhibit different intrinsic twists and torsion and bending elastic constants. Expressions are derived to relate the effective ET and l0 to the equilibrium constant, water activity (aw), and number (n) of bound water molecules released per cooperative domain undergoing the two-state transition. The variations of l0 and ET with -ln(aw) are similar for acetamide and ethylene glycol at 37 degrees C. Fitting the theory to those data yields the range n = 103-125 for ethylene glycol and n = 71-113 for acetamide, depending on the assumed value of ET for the dehydrated state. The cooperative domain size of the two-state transition is estimated to exceed about 25-30 base pairs (bp). Between 0 and 19.4 w/v % ethylene glycol, the torsion elastic constant, measured by time-resolved fluorescence polarization anisotropy (FPA), increases by 1.37-fold, whereas the measured ET decreases by 1.15-fold over that same range. The implied decrease in bending rigidity over that range is by a factor of about 0.7. The variations of l0 and ET with increasing -ln(aw) due to added ethylene glycol at 37 degrees C are far smaller than the corresponding variations observed previously at 14 and 15 degrees C. However, at 21 degrees C, upon adding either ethylene glycol or acetamide, l0 and ET initially decline steeply with increasing -ln(aw), with slopes possibly comparable to those seen at 14 and 15 degrees C, but then flatten out and follow curves similar to those at 37 degrees C. Possible origins of such mixed behavior are discussed. The effects of betaine at both 37 and 21 degrees C differ qualitatively and quantitatively in various respects from those of ethylene glycol and acetamide. Upon adding sucrose, l0 initially jumps to higher plateaus at both 37 and 21 degrees C, but its effects on ET cannot be reliably assessed, due to the limited range of -ln(aw).     

Tensorial elastic network model for protein dynamics: Integration of the anisotropic network model with bond‐bending and twist elasticities

We present a tensorial elastic network model (TNM) to describe the equilibrium fluctuations of proteins near their native fold structure. The model combines the anisotropic network model (ANM), bond bending elasticity, and backbone twist elasticity, and can predict both the isotropic fluctuations, similar to the Gaussian network model (GNM), and anisotropic fluctuations, similar to the ANM. TNM performs equally well for B‐factor predictions as GNM and predicts the anisotropy of B‐factors better than ANM. The model also outperforms the ANM in its predictability of the complete anisotropic displacement parameters. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

To twist or not to twist: From chromophore structure to dynamics inside engineered photoconvertible and photoswitchable fluorescent proteins

Green-to-red photoconvertible fluorescent proteins (FPs) are vital biomimetic tools for powerful techniques such as super-resolution imaging. A unique Kaede-type FP named the least evolved ancestor (LEA) enables delineation of the evolutionary step to acquire photoconversion capability from the ancestral green fluorescent protein (GFP). A key residue, Ala69, was identified through several steady-state and time-resolved spectroscopic techniques that allows LEA to effectively photoswitch and enhance the green-to-red photoconversion. However, the inner workings of this functional protein have remained elusive due to practical challenges of capturing the photoexcited chromophore motions in real time. Here, we implemented femtosecond stimulated Raman spectroscopy and transient absorption on LEA-A69T, aided by relevant crystal structures and control FPs, revealing that Thr69 promotes a stronger π–π stacking interaction between the chromophore phenolate (P-)ring and His193 in FP mutants that cannot photoconvert or photoswitch. Characteristic time constants of ~60–67 ps are attributed to P-ring twist as the onset for photoswitching in LEA (major) and LEA-A69T (minor) with photoconversion capability, different from ~16/29 ps in correlation with the Gln62/His62 side-chain twist in ALL-GFP/ALL-Q62H, indicative of the light-induced conformational relaxation preferences in various local environments. A minor subpopulation of LEA-A69T capable of positive photoswitching was revealed by time-resolved electronic spectroscopies with targeted light irradiation wavelengths. The unveiled chromophore structure and dynamics inside engineered FPs in an aqueous buffer solution can be generalized to improve other green-to-red photoconvertible FPs from the bottom up for deeper biophysics with molecular biology insights and powerful bioimaging advances.     

Structural and sequence features of two residue turns in beta‐hairpins

Beta‐turns in beta‐hairpins have been implicated as important sites in protein folding. In particular, two residue β‐turns, the most abundant connecting elements in beta‐hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta‐hairpins with a large data set. For this, 3977 beta‐turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta‐hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two‐residue turns, and their distributions were examined. From this study, we could identify code‐like sequence motifs for the two residue beta‐turn types. Finally, structural and sequence properties of beta‐strands in the beta‐hairpins were analyzed, which revealed that the beta‐strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta‐hairpin turn structures and sequences. Proteins 2014; 82:1721–1733. © 2014 Wiley Periodicals, Inc.     

A Maverick Asymmetrical Backbone with Distinct Flanked Twist Angles Modulating the Molecular Aggregation and Crystallinity for High Performance Nonfullerene Solar Cells

In this work, a new asymmetrical backbone thienobenzodithiophene (TBD) containing four aromatic rings is designed, and then four polymers PTBD‐BZ, PTBD‐BDD, PTBD‐FBT, and PTBD‐Tz are synthesized. The planar and high degree of π‐conjugation configuration can guarantee effective charge carrier transport and the distinct flanked dihedral angles between the TBD core and conjugated side chain can subtly regulate the molecular aggregation and crystallinity. The four polymer/3,9‐bis(2‐methylene‐(3‐(1,1‐dicyanomethylene)‐indanone)‐5,5,11,11‐tetrakis(4‐hexylphenyl)‐dithieno[2,3‐d:2′,3′‐d′]‐s‐indaceno[1,2‐b:5,6‐b′]‐dithiophene (ITIC) blending films exhibit predominantly face‐on orientation. The photovoltaic devices based on wide bandgap polymers PTBD‐BZ and PTBD‐BDD achieve power conversion efficiencies (PCEs) as high as 12.02% and 11.39% without any post‐treatment. For the medium bandgap polymers PTBD‐FBT and PTBD‐Tz, the devices also show good PCEs of 10.18% and 11.02% with high V_OC of 0.94 and 1.02 V, respectively, which indicates simultaneously achieving a V_OC > 1 V and a high J_SC is feasible to further improve the PSCs' performance by modifying this new backbone. This work reveals that the versatile asymmetric backbone is an excellent moiety to construct light‐harvesting copolymers and to modulate the microstructure for highly efficient PSCs.     

The Hox Transcription Factor Ubx Ensures Somatic Myogenesis by Suppressing the Mesodermal Master Regulator Twist

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