The byssus of the zebra mussel (Dreissena polymorpha): spatial variations in protein composition

The notorious biofouling organism Dreissena polymorpha (the zebra mussel) attaches to a variety of surfaces using a byssus, a series of protein threads that connect the animal to adhesive plaques secreted onto hard substrata. Here, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the composition of different regions of the byssus is reported. All parts of the byssus show mass peaks corresponding to small proteins in the range of 3.7–7 kDa, with distinctive differences between different regions. Indeed, spectra from thread and plaques are almost completely non-overlapping. In addition, several peaks were identified that are unique to the interfacial region of the plaque, and therefore likely represent specialized adhesive proteins. These results indicate a high level of control over the distribution of proteins, presumably with different functions, in the byssus of this freshwater species.     

The Genetic Algorithm and the Conformational Search of Polypeptides and Proteins

Abstract

The genetic algorithm is a technique of function optimization derived from the principles of evolutionary theory. We have adapted it to perform conformational search on polypeptides and proteins. The algorithm was first tested on several small polypeptides and the 46 amino acid protein crambin under the AMBER potential energy function. The probable global minimum conformations of the polypeptides were located 90% of the time and a non-native conformation of crambin was located that was 150kcal/mol lower in potential energy than the minimized crystal structure conformation. Next, we used a knowledge-based potential function to predict the structures of melittin, pancreatic polypeptide, and crambin. A 2.31 Å ΔRMS conformation of melittin and a 5.33 Å ΔRMS conformation of pancreatic polypeptide were located by genetic algorithm-based conformational search under the knowledge-based potential function. Although the ΔRMS of pancreatic polypeptide was somewhat high, most of the secondary structure was correct. The secondary structure of crambin was predicted correctly, but the potential failed to promote packing interactions. Finally, we tested the packing aspects of our potential function by attempting to predict the tertiary structure of cytochrome b ₅₆₂ given correct secondary structure as a constraint. The final predicted conformation of cytochrome b ₅₆₂ was an almost completely extended continuous helix which indicated that the knowledge-based potential was useless for tertiary structure prediction. This work serves as a warning against testing potential functions designed for tertiary structure prediction on small proteins.     

Immunoglobulin immobilized liposomal constructs for transmucosal vaccination through nasal route

The aim of the present investigation was to evaluate the prospective of surface-engineered vesicular carriers for mucosal immunization via the nasal route. IgG antibody was immobilized on the surface of hepatitis B surface antigen (HBsAg) antigen–loaded liposomes. The developed formulations were characterized on the basis of physicochemical parameters, such as morphology, particle size, polydispersity index, entrapment efficiency, and zeta potential. Liposomal formulations were then evaluated for in-process antigen stability and storage stability. In vivo studies were conducted to visualize targeting potential, localization pattern, and immunogenicity. In addition, immune response was compared with alum-HBsAg vaccine injected intramuscularly. The serum anti-HBsAg titer, obtained from the postnasal administration of IgG-coupled liposomes, was significantly higher than plain liposomes. Moreover, IgG-coupled liposomes generated both humoral (i.e., systemic and mucosal) and cellular immune responses upon nasal administration, while the alum-adsorbed antigen displayed neither cellular (cytokine level) nor mucosal (IgA) response. The formulation also displayed enhanced transmucosal transport, improved in vitro stability, and effective immunoadjuvant property. To conclude, IgG antibody-coupled liposomes may serve as novel carriers to augment the secretory immune response of antigen encapsulated in the liposomes, apparently by escalating liposome uptake via M cells, thereby rationalizing their use as a carrier adjuvant for nasal subunit vaccines.     

Density discriminates between thermophilic and mesophilic proteins

Despite an intense interest and a remarkable number of studies on the subject, the relationships between thermostability and (primary, secondary and tertiary) structure of proteins are still not fully understood. Here, comparing the protein density – defined by the ratio between the residue number and protein excluded volume – for a set of thermophilic/mesophilic pairs, we provide evidence that this property is connected to the optimal growth temperature. In particular, our results indicate that thermophilic proteins have – in general – a lower density with respect to the mesophilic counterparts, being such a correlation more pronounced for optimal growth temperature differences greater than 40°C. The effect of the protein thermostability changes on the molecular shape is also presented.     

Macromolecular Design,Nucleic Acid Junctions,and Crystal Formation

Abstract

The simplest form of macromolecular design involves the ligation of nucleic acids. Recent results on the concatenation of nucleic acid junctions show that these molecules can act as fairly rigid macromolecular valence clusters on the nanometer scale. These clusters can be joined to form closed stick figures in which each edge is double helical DNA or RNA and each vertex is a nucleic acid junction. The geometrical criteria for forming discrete-closed and periodic structures from these components are established. The helicity of each edge limits the possible structures that can be formed.

The formation of a periodic array from nucleic acid junction building blocks is compared with the crystallization of molecular systems. This comparison leads to a new interpretation of the nature of order in the solid state for molecular crystals. The suggestion is made that the structure of a solid molecular system described by the fewest unique orthogonal (Fourier) components is the one which will be entropically favored, since it contains the least information. This is the crystalline state, with a small number of molecules per asymmetric unit. The free energy from the proposed entropie driving force responsible for this behavior is available, in principle, to correct small deviations from ideality in forming covalent crystals from nucleic acid junction components, as well as in non-bonded molecular systems. Nucleic acid junction periodic arrays provide an appropriate vehicle with which to test this interpretation.     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Insights into the structural stability of nuclear matrix ribonucleoprotein,LMG160: thermodynamic and spectroscopic analysis

Low-mobility group nonhistone chromatin protein, LMG_160, is a nuclear matrix ribonucleoprotein particle (RNP) which has a RNA molecule with approximately 300 bases. In this study, structural stability of the intact LMG₁₆₀ (I-LMG₁₆₀) was investigated at different ionic strength and in the absence of its RNA moiety (T-LMG₁₆₀) employing spectroscopic and thermodynamic techniques. The UV absorption spectra showed hypochromicity and red shift under increasing ionic strength for both forms of LMG₁₆₀ but in different extents. The fluorescence emission intensity was decreased as ionic strength was increased and the Stern–Volmer quenching constant (K_sv) for T-LMG₁₆₀ was 3.7 times less than for I-LMG₁₆₀. In the absence of sodium chloride, I-LMG₁₆₀ exhibited a very stable structure against the temperature change compared to T-LMG₁₆₀. The thermodynamic parameters showed that the positive values of ΔH_m and ΔS_m increased by increasing ionic strength in both forms of LMG₁₆₀. Removal of the RNA moiety altered secondary structure: as T-LMG₁₆₀ showed more helical content than I-LMG₁₆₀. From the results, it is concluded that I-LMG₁₆₀ is more sensitive to alteration of environment and the RNA has an important role in this RNP conformation. Also, interaction of both I- and T-LMG₁₆₀ with sodium chloride is entropy driven and is usually accompanied by surface hydrophobicity.     

An N-terminal, 830 residues intrinsically disordered region of the cytoskeleton-regulatory protein supervillin contains Myosin II- and F-actin-binding sites

Supervillin, the largest member of the villin/gelsolin family, is a cytoskeleton regulating, peripheral membrane protein. Supervillin increases cell motility and promotes invasive activity in tumors. Major cytoskeletal interactors, including filamentous actin and myosin II, bind within the unique supervillin amino terminus, amino acids 1–830. The structural features of this key region of the supervillin polypeptide are unknown. Here, we utilize circular dichroism and bioinformatics sequence analysis to demonstrate that the N-terminal part of supervillin forms an extended intrinsically disordered region (IDR). Our combined data indicate that the N-terminus of human and bovine supervillin sequences (positions 1–830) represents an IDR, which is the largest IDR known to date in the villin/gelsolin family. Moreover, this result suggests a potentially novel mechanism of regulation of myosin II and F-actin via the intrinsically disordered N-terminal region of hub protein supervillin.     

Enhanced sampling of molecular dynamics simulation of peptides and proteins by double coupling to thermal bath

Low sampling efficiency in conformational space is the well-known problem for conventional molecular dynamics. It greatly increases the difficulty for molecules to find the transition path to native state, and costs amount of CPU time. To accelerate the sampling, in this paper, we re-couple the critical degrees of freedom in the molecule to environment temperature, like dihedrals in generalized coordinates or nonhydrogen atoms in Cartesian coordinate. After applying to ALA dipeptide model, we find that this modified molecular dynamics greatly enhances the sampling behavior in the conformational space and provides more information about the state-to-state transition, while conventional molecular dynamics fails to do so. Moreover, from the results of 16 independent 100?ns simulations by the new method, it shows that trpzip2 has one-half chances to reach the naive state in all the trajectories, which is greatly higher than conventional molecular dynamics. Such an improvement would provide a potential way for searching the conformational space or predicting the most stable states of peptides and proteins.     

Hydrolases: The Correlation Between Informational Structure and the Catalytic Sites Organization

Abstract

The novel method allowing identification of protein structure elements responsible for catalytic activity manifestation is proposed. Structural organization of various hydrolases was studied using the ANIS (ANalysis of Informational Structure) method. ANIS allows to reveal a hierarchy of the ELements of Information Structure (ELIS) using protein amino acid sequence. The ELIS corresponds to the variable length sites with an increased density of structural information. The amino acid residues forming the enzyme catalytic site were shown to belong to the different top-ranking ELIS located in the contact area of the corresponding spatial structure clusters. In the protein spatial structure catalytic sites are located in the area of contact between fragments of polypeptide chain (structural blocs) allocation to the different top-ranking ELIS. According to our results we concluded that structural blocks corresponding to top-ranking ELIS are crucial for protein functioning. Such regions are structurally independent, and their determinate mobility relative to each other is vital for an efficient enzymatic reaction to occur.