FtsZ condensates: An in vitro electron microscopy study

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto‐filaments. Although FtsZ assembles into single proto‐filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z‐ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 340–350, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Mg2+ ions bind at the C‐terminal region of skeletal muscle α‐tropomyosin

Tropomyosin (Tm) is a dimeric coiled‐coil protein that polymerizes through head‐to‐tail interactions. These polymers bind along actin filaments and play an important role in the regulation of muscle contraction. Analysis of its primary structure shows that Tm is rich in acidic residues, which are clustered along the molecule and may form sites for divalent cation binding. In a previous study, we showed that the Mg²⁺‐induced increase in stability of the C‐terminal half of Tm is sensitive to mutations near the C‐terminus. In the present report, we study the interaction between Mg²⁺ and full‐length Tm and smaller fragments corresponding to the last 65 and 26 Tm residues. Although the smaller Tm peptide (Tm_{259‐284(W269)}) is flexible and to large extent unstructured, the larger Tm_{220‐284(W269)} fragment forms a coiled coil in solution whose stability increases significantly in the presence of Mg²⁺. NMR analysis shows that Mg²⁺ induces chemical shift perturbations in both Tm_{220‐284(W269)} and Tm_{259‐284(W269)} in the vicinity of His276, in which are located several negatively charged residues. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 583–590, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Direct real‐time imaging of protein adsorption onto hydrophilic and hydrophobic surfaces

Atomic force microscopy has been used to follow in real time the adsorption from solution of two of the gliadin group of wheat seed storage proteins onto hydrophilic (mica) and hydrophobic (graphite) surfaces. The liquid cell of the microscope was used initially to acquire images of the substrate under a small quantity of pure solvent (1% acetic acid). Continuous imaging as an injection of gliadin solution entered the liquid cell enabled the adsorption process to be followed in situ from zero time. For ω‐gliadin, a monolayer was formed on the mica substrate during a period of ～2000 s, with the protein molecules oriented in parallel to the mica surface. In contrast, the ω‐gliadin had a relatively low affinity for the graphite substrate, as demonstrated by slow and weak adsorption to the surface. With γ‐gliadin, random deposition onto the mica surface was observed forming monodispersed structures, whereas on the graphite surface, monolayer islands of protein were formed with the protein molecules in a perpendicular orientation. Sequential adsorption experiments indicated strong interactions between the two proteins that, under certain circumstances, caused alterations to the surface morphologies of preadsorbed species. The results are relevant to our understanding of the interactions of proteins within the hydrated protein bodies of wheat grain and how these determine the processing properties of wheat gluten and dough. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 74–84, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The immunosuppressive activity and solution structures of ubiquitin fragments

Recently, ubiquitin was suggested as a promising anti‐inflammatory protein therapeutic. We found that a peptide fragment corresponding to the ubiquitin^50–59 sequence (LEDGRTLSDY) possessed the immunosuppressive activity comparable with that of ubiquitin. CD and NMR spectroscopies were used to determine the conformational preferences of LEDGRTLSDY in solution. The peptide mixture, obtained by pepsin digestion of ubiquitin, was even more potent than the intact protein. Although the peptide exhibited a well‐defined conformation in methanol, its structure was distinct from the corresponding 50–59 fragment in the native ubiquitin molecule. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 423–431, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Changes in the quaternary structure of amelogenin when adsorbed onto surfaces

Amelogenin is a unique protein that self‐assembles into spherical aggregates called “nanospheres” and is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin onto substrates is of great interest because protein‐surface interactions are critical to its function. We report studies of the adsorption of amelogenin onto self‐assembled monolayers containing COOH end group functionality as well as single crystal fluoroapatite, a biologically relevant surface. We found that although our solutions contained only nanospheres of narrow size distribution, smaller structures such as dimers or trimers were observed on the hydrophilic surfaces. This suggests that amelogenin can adsorb onto surfaces as small structures that “shed” or disassemble from the nanospheres that are present in solution. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 103–107, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Ultrastructure of ulvan: A polysaccharide from green seaweeds

Ultrastructural analysis of the gel forming green seaweed sulfated polysaccharide ulvan revealed a spherical‐based morphology (10–18 nm diameter) more or less aggregated in aqueous solution. At pH 13 in TBAOH (tetrabutyl ammonium hydroxyde) or NaOH, ulvan formed an open gel‐like structure or a continuous film by fusion or coalescence of bead‐like structures, while in acidic pH conditions, ulvan appeared as dispersed beads. Low concentrations of sodium chloride, copper or boric acid induced the formation of aggregates. These results highlight the hydrophobic and aggregative behavior of ulvan that are discussed in regard to the peculiar gel formation and the low intrinsic viscosity of the polysaccharide in aqueous solution. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 652–664, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Peptidic modulators of protein‐protein interactions: Progress and challenges in computational design

With the decline in productivity of drug‐development efforts, novel approaches to rational drug design are being introduced and developed. Naturally occurring and synthetic peptides are emerging as novel promising compounds that can specifically and efficiently modulate signaling pathways in vitro and in vivo. We describe sequence‐based approaches that use peptides to mimic proteins in order to inhibit the interaction of the mimicked protein with its partners. We then discuss a structure‐based approach, in which protein‐peptide complex structures are used to rationally design and optimize peptidic inhibitors. We survey flexible peptide docking techniques and discuss current challenges and future directions in the rational design of peptidic inhibitors. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 505–513, 2009. This article was originally published online as an accepted preprint. The “Published Online”date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Importance of the spatial display of charged residues in heparin–peptide interactions

Many studies have examined consensus sequences required for protein‐glycosaminoglycan interactions. Through the synthesis of helical heparin binding peptides, this study probes the relationship between spatial arrangement of positive charge and heparin binding affinity. Peptides with a linear distribution of positive charge along one face of the α‐helix had the highest affinity for heparin. Moving the basic residues away from a single face resulted in drastic changes in heparin binding affinity of up to three orders of magnitude. These findings demonstrate that amino acid sequences, different from the known heparin binding consensus sequences, will form high affinity protein‐heparin binding interactions when the charged residues are aligned linearly. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 290–298, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Hsp70 molecular chaperones and Parkinson's disease

Because over expression of Hsp70 molecular chaperones suppresses the toxicity of aberrantly folded proteins that occur in Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and various polyQ‐diseases (Huntington's disease and ataxias), Hsp70 is garnering attention as a possible therapeutic agent for these various diseases. Here, I review progress in this fascinating field of molecular chaperones and neurodegeneration and describe our current understanding of the mechanisms by which Hsp70 protects cells from the PD‐related protein called alpha‐synuclein (α‐syn). © 2009 Wiley Periodicals, Inc. Biopolymers 93: 218–228, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Adhesion of pancreatic beta cells to biopolymer films

Dramatic reversal of Type 1 diabetes in patients receiving pancreatic islet transplants continues to prompt vigorous research concerning the basic mechanisms underlying patient turnaround. At the most fundamental level, transplanted islets must maintain viability and function in vitro and in vivo and should be protected from host immune rejection. Our previous reports showed enhancement of islet viability and insulin secretion per tissue mass for small islets (<125 μm) as compared with large islets (>125 μm), thus, demonstrating the effect of enhancing the mass transport of islets (i.e. increasing tissue surface area to volume ratio). Here, we report the facile dispersion of rat islets into individual cells that are layered onto the surface of a biopolymer film towards the ultimate goal of improving mass transport in islet tissue. The tightly packed structure of intact islets was disrupted by incubating in calcium‐free media resulting in fragmented islets, which were further dispersed into individual or small groups of cells by using a low concentration of papain. The dispersed cells were screened for adhesion to a range of biopolymers and the nature of cell adhesion was characterized for selected groups by quantifying adherent cells, measuring the surface area coverage of the cells, and immunolabeling cells for adhesion proteins interacting with selected biopolymers. Finally, beta cells in suspension were centrifuged to form controlled numbers of cell layers on films for future work determining the mass transport limitations in the adhered tissue constructs. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 676–685, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Thermodynamically controlled supramolecular polymerization of cytochrome b562

A hemoprotein‐based supramolecular polymer that has a covalently linked heme moiety on the protein surface has been constructed based on interprotein heme–heme pocket interactions of the chemically modified apocytochrome b₅₆₂ ( 1 ‐H63C). The thermodynamic properties of the polymer have been investigated by means of size exclusion chromatography, UV–vis spectroscopy, and circular dichroism spectroscopy. The results indicate that, as with other synthetic systems reported so far, the 1 ‐H63C hemoprotein assembly is thermodynamically controlled in aqueous solution: the degree of polymerization is dependent on the 1 ‐H63C concentration and is modulated by the addition of the end‐capping units, native heme, and/or apocytochrome b₅₆₂ mutant (apoH63C). These properties suggest a potential use for the hemoprotein self‐assembly in preparation of stimuli‐responsive functional nanobiomaterials. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 194–200, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Kinetics of the self‐aggregation and film formation of poly‐L‐proline at high temperatures explored by circular dichroism spectroscopy

Poly‐L ‐proline has been used as a model system for various purposes over a period of more than 60 years. Its relevance among the protein/peptide community stems from its use as a reference system for determining the conformational distributions of unfolded peptides and proteins, its use as a molecular ruler, and from the pivotal role of proline residues in conformational transitions and protein–protein interactions. While several studies indicate that polyproline can aggregate and precipitate in aqueous solution, a systematic study of the aggregation process is still outstanding. We found, by means of UV‐circular dichroism and IR measurements, that polyproline is predominately monomeric at room temperature at millimolar concentrations. Upon heating, the polypeptide stays in its monomeric state until the temperature reaches a threshold of ca. 60°C. At higher temperatures, the peptide aggregates as a film on the inside surface of the employed cuvette. The process proceeds on a time scale of 10³ s and can best be described by a bi‐exponential relaxation function. The respective CD and IR spectra are qualitatively different from the canonical spectra of polyproline in aqueous solution, and are indicative of a highly packed state. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 451–457, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Probing nanostructures of bacterial extracellular polymeric substances versus culture time by Raman microspectroscopy and atomic force microscopy

The structure of a bacterial cell wall may alter during bacterial reproduction. Moreover, these cell wall variations, on a nanoscale resolution, have not yet fully been elucidated. In this work, Raman spectroscopy and atomic force microscopy (AFM) technique are applied to evaluate the culture time‐dependent cell wall structure variations of Pseudomonas putida KT2440 at a quorum and single cell level. The Raman spectra indicate that the appearance of DNA/RNA, protein, lipid, and carbohydrates occurs till 6 h of cultivation time under our experimental conditions. AFM characterization reveals the changes of the cellular surface ultrastructures over the culture time period, which is a gradual increase in surface roughness during the time between the first two and eight hours cultivation time. This work demonstrates the feasibility of utilizing a combined Raman spectroscopy and AFM technique to investigate the cultivation time dependence of bacterial cellular surface biopolymers at single cell level. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 171–177, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Hairpin ribozyme catalysis: A surface‐enhanced Raman spectroscopy study

The existence of an “RNA world” as an early step in the history of life increases the interest for the characterization of these biomolecules. The hairpin ribozyme studied here is a self‐cleaving/ligating motif found in the minus strand of the satellite RNA associated with Tobacco ringspot virus. Surface‐enhanced Raman spectroscopy (SERS) is a powerful tool to study trace amounts of RNA. In controlled conditions, a SERS signal is proportional to the amount of free residues adsorbed on the metal surface. On RNA cleavage, residues are unpaired and free to interact with metal. SERS procedures are used to monitor and quantify the catalysis of ribozyme cleavage at biological concentrations in real time; thus, they propose an interesting alternative to electrophoretic methods. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 384–390, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Rapid determination of antigenic epitopes in human NGAL using NMR

The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two‐dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high‐affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase‐associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in ¹H‐¹⁵N‐TROSY HSQC and ¹H‐¹³C HSQC spectra of isotope‐labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual‐color fluorescence cross‐correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel‐like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR‐based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 657–667, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Partition of thermodynamic energies of drug–DNA complexation

We report a computation methodology, which leads to the ability to partition the Gibb's free energy for the complexation reaction of aromatic drug molecules with DNA. Using this approach, it is now possible to calculate the absolute values of the energy contributions of various physical factors to the DNA binding process, whose summation gives a value that is reasonably close to the experimentally measured Gibb's free energy of binding. Application of the methodology to binding of various aromatic drugs with DNA provides an answer to the question “What forces are the main contributors to the stabilization of aromatic ligand–DNA complexes?” © 2009 Wiley Periodicals, Inc. Biopolymers 91: 773–790, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Revisiting “reverse hydrophobic effect”: Applicable only to coil mutations at the surface

In a seminal paper, Pakula and Sauer (Nature, 1990, 344, 363–364) demonstrated that the increase in side‐chain hydrophobicity has a reverse relationship with protein stability. We have addressed this problem with several examples of mutants that span at different locations in protein structure based on secondary structure and solvent accessibility. We confirmed that the stability change upon single coil mutation at exposed region is reversely correlated with hydrophobicity with a single exception. In addition, we found the existence of such relationship in partially buried coil mutants. The stability of exposed helical mutants is governed by conformational properties. In buried and partially buried helical and strand mutants properties reflecting hydrophobicity have direct relationship with stability, whereas an opposite relationship was obtained with entropy and flexibility. The structural analysis of partially buried/exposed mutants showed that the surrounding residues are important for the stability change upon mutation. These results provide insights to understand the general behavior for the stability of proteins upon amino acid substitutions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 591–599, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Electrostatic interactions with histone tails may bend linker DNA in chromatin

Is linker DNA bent in the 30‐nm chromatin fiber at physiological conditions? We show here that electrostatic interactions between linker DNA and histone tails including salt condensation and release may bend linker DNA, thus affecting the higher order organization of chromatin. © 2005 Wiley Periodicals, Inc. Biopolymers 81: 20–28, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Spectroscopic and molecular modeling studies on the binding of the flavonoid luteolin and human serum albumin

Luteolin (LUT) is a polyphenolic compound, found in a variety of fruits, vegetables, and seeds, which has a variety of pharmacological properties. In the present contribution, binding of LUT to human serum albumin (HSA), the most abundant carrier protein in the blood, was investigated with the aim of describing the binding mode and parameters of the interaction. The application of circular dichroism, UV‐Vis absorption, fluorescence, Raman and surface‐enhanced Raman scattering spectroscopy combined with molecular modeling afforded a clear picture of the association mode of LUT to HSA. Specific interactions with protein amino acids were evidenced. LUT was found to be associated in subdomain IIA where an interaction with Trp‐214 is established. Hydrophobic and electrostatic interactions are the major acting forces in the binding of LUT to HSA. The HSA conformations were slightly altered by the drug complexation with reduction of α‐helix and increase of β‐turns structures, suggesting a partial protein unfolding. Also the configuration of at least two disulfide bridges were altered. Furthermore, the study of molecular modeling afforded the binding geometry. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 917–927, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com