Large Biological Molecules Research Articles

Surface‐enhanced Raman scattering of 4,4'‐diaminostilbene: Dependence of spectral features and resonant enhancement on surface coverage

AbstractAt any applications of surface‐enhanced Raman scattering (SERS) spectroscopy, from detection of small ions to recognition of large biological molecules, an understanding the spectral profiles and its transformation occurring under changes of local environment is an important stage essential for a reliable chemical analysis. In this work, we showed that the adsorption of 4,4'‐diaminostilbene on Ag nanoparticles is accompanied by a dramatic change in the SERS profile observed at transition from submonolayer to multilayer coverage. A profile deconvolution analysis allowed revealing the spectral signatures inherent for the surface species existing at submonolayer and multilayer adsorption corresponding to the hot spots and ordinary conditions, respectively. A resonance character of Raman enhancement was detected in the case of 4,4'‐diaminostilbene adsorbed in hot spots regions. A feasibility of ligand to metal charge transfer was proved on the basis of ultraviolet–visible data. The overtones and combination bands were observed in the SERS spectra at resonance conditions. The spectral signatures of the molecules inside and outside hot spots regions were assigned. A considerable activation of rings vibrations together with a suppression of ethylenic bridge vibrations was revealed in both cases. The most prominent features of 4,4'‐diaminostilbene linked with two Ag nanoparticles in the range 200–1,800 cm‐1 are the medium bands at 581 and 1249 cm‐1 corresponding to CCC bending and C–N stretching modes, respectively. The obtained results can serve as a basis for development of new powerful Raman techniques for evaluation of surface coverage and “hot spots mapping,” which are of great interest for surface science and plasmonics.

Challenges and advances in atomistic simulations of potassium and sodium ion channel gating and permeation.

Ion channels are implicated in many essential physiological events such as electrical signal propagation and cellular communication. The advent of K+ and Na+ ion channel structure determination has facilitated numerous investigations of molecular determinants of their behaviour. At the same time, rapid development of computer hardware and molecular simulation methodologies has made computational studies of large biological molecules in all-atom representation tractable. The concurrent evolution of experimental structural biology with biomolecular computer modelling has yielded mechanistic details of fundamental processes unavailable through experiments alone, such as ion conduction and ion channel gating. This review is a short survey of the atomistic computational investigations of K+ and Na+ ion channels, focusing on KcsA and several voltage-gated channels from the KV and NaV families, which have garnered many successes and engendered several long-standing controversies regarding the nature of their structure-function relationship. We review the latest advancements and challenges facing the field of molecular modelling and simulation regarding the structural and energetic determinants of ion channel function and their agreement with experimental observations.

Achieving Stable Electrospray Ionization Mass Spectrometry Detection from Microfluidic Chips.

The past two decades have witnessed remarkable advances in the development of microfluidic devices as bioanalytical platforms for the analysis of biological molecules. The implementation of mass spectrometry (MS) detection systems on these devices has become inevitable, and various chip-MS ionization interfaces have been developed. As electrospray ionization (ESI) is particularly relevant for the analysis of large biological molecules such as proteins or peptides, efforts have focused on advancing interfaces that meet the demands of nano-separation techniques that are typically used prior to MS detection. Achieving stable ESI conditions that enable sensitive MS detection is, however, not trivial, especially when the spray is generated from a microfabricated platform. This chapter is aimed at providing a step-by-step protocol for producing stable and efficient electrospray sample ionization from microfluidic chips that are used for capillary electrophoresis (CE) separations.

IMass Time: The Future, in Future!

Joseph John Thomson discovered and proved the existence of electrons through a series of experiments. His work earned him a Nobel Prize in 1906 and initiated the era of mass spectrometry (MS). In the intervening time, other researchers have also been awarded the Nobel Prize for significant advances in MS technology. The development of soft ionization techniques was central to the application of MS to large biological molecules and led to an unprecedented interest in the study of biomolecules such as proteins (proteomics), metabolites (metabolomics), carbohydrates (glycomics), and lipids (lipidomics), allowing a better understanding of the molecular underpinnings of health and disease. The interest in large molecules drove improvements in MS resolution and now the challenge is in data deconvolution, intelligent exploitation of heterogeneous data, and interpretation, all of which can be ameliorated with a proposed IMass technology. We define IMass as a combination of MS and artificial intelligence, with each performing a specific role. IMass will offer advantages such as improving speed, sensitivity, and analyses of large data that are presently not possible with MS alone. In this study, we present an overview of the MS considering historical perspectives and applications, challenges, as well as insightful highlights of IMass.

Mesoscopic Rigid Body Modelling of the Extracellular Matrix Self-Assembly.

The extracellular matrix (ECM) plays an important role in supporting tissues and organs. It even has a functional role in morphogenesis and differentiation by acting as a source of active molecules (matrikines). Many diseases are linked to dysfunction of ECM components and fragments or changes in their structures. As such it is a prime target for drugs. Because of technological limitations for observations at mesoscopic scales, the precise structural organisation of the ECM is not well-known, with sparse or fuzzy experimental observables. Based on the Unity3D game and physics engines, along with rigid body dynamics, we propose a virtual sandbox to model large biological molecules as dynamic chains of rigid bodies interacting together to gain insight into ECM components behaviour in the mesoscopic range. We have preliminary results showing how parameters such as fibre flexibility or the nature and number of interactions between molecules can induce different structures in the basement membrane. Using the Unity3D game engine and virtual reality headset coupled with haptic controllers, we immerse the user inside the corresponding simulation. Untrained users are able to navigate a complex virtual sandbox crowded with large biomolecules models in a matter of seconds.

Physical Methods for Determination of Biomolecular Structures

We have described various physical methods, which have been employed in the determination of three dimensional structures of biological molecules. The basic principles of methods currently in use, have been described. Principles of two major techniques, namely NMR and X-ray crystallography have been discussed in detail. Applications of such techniques, to study large biological molecules and intermolecular interactions between such molecules have been described. Molecular assemblies, containing more than one molecule and complex structures such as ribosomes have been discussed.

N₂-BET is a Proxy for Primary Particle Size and May Not Be Representative of Available Specific Surface Area for Aggregated Nanoparticle Aerosols.

The knowledge of the specific surface area of aerosolized engineered nanoparticles could be important for mechanistically understanding their toxic potential or functional characteristics. The most widely method to perform this measurement, N2-BET, however, may not accurately represent the available surface area for hetero-aggregated nanoparticles in the context of large biological molecules. This study conducted an analysis of published characterization measurements including primary particle size, aggregation state, and specific surface area made for dry aerosolized nanoparticles. Results indicate that primary particle size explains 65% of the variance in specific surface area, while aggregation (as measured by mass median aerodynamic diameter) only explains 20% of the variance. Curiously, increasing aggregation (larger MMAD) is associated with increasing SSA as measured by N2-BET, likely an artifact of the measurement method, which suggests that this technique may not be appropriate for studies investigating biological interactions with nanoparticles.

Laser postionization of neutral molecules sputtered using bismuth and argon cluster primary ions.

In this study, the influence of two different cluster primary ions in laser secondary neutral mass spectrometry (Laser-SNMS) has been investigated. Despite the many advantages of Laser-SNMS, fragmentation of neutral organic molecules during both sputtering and photoionization has limited its efficiency for the study of large organic and biological molecules. Cluster ion sputtering, and in particular large argon gas cluster sputtering, has been proposed as a means of reducing this fragmentation. Molecules of 9-fluorenylmethoxycarbonyl-pentafluoro-l-phenylalanine were sputtered using Bi3+ and Ar2000+ cluster primary ions, and the desorbed neutral species ("secondary neutrals") were postionized using a 7.87 eV vacuum ultraviolet laser light fluorine excimer laser. By varying timing parameters and laser power density, time-of-flight and laser power density distributions were obtained to investigate the fragmentation and energy distributions of the sputtered neutral species. Changing from 30 keV Bi3+ sputtering to 10 keV Ar2000+ resulted in a significant reduction in fragmentation of the molecule as well as a suppression of the high background that results from metastable decay of highly excited ions, yielding significantly improved detection of the intact molecule and characteristic fragments. Analysis of the influence of laser power density and laser pulse delay time indicates a reduction of fragmentation in both the sputtering phase and the photoionization phase. This study demonstrates the importance of soft desorption for efficient laser postionization of large organic molecules and shows the potential for improving the efficiency of laser postionization by using large gas cluster ion sputtering.

Molecular mechanism of water reorientational slowing down in concentrated ionic solutions

Water dynamics in concentrated ionic solutions plays an important role in a number of material and energy conversion processes such as the charge transfer at the electrolyte-electrode interface in aqueous rechargeable ion batteries. One long-standing puzzle is that all electrolytes, regardless of their "structure-making/breaking" nature, make water rotate slower at high concentrations. To understand this effect, we present a theoretical simulation study of the reorientational motion of water molecules in different ionic solutions. Using an extended Ivanov model, water rotation is decomposed into contributions from large-amplitude angular jumps and a slower frame motion which was studied in a coarse-grained manner. Bearing a certain resemblance to water rotation near large biological molecules, the general deceleration is found to be largely due to the coupling of the slow, collective component of water rotation with the motion of large hydrated ion clusters ubiquitously existing in the concentrated ionic solutions. This finding is at variance with the intuitive expectation that the slowing down is caused by the change in fast, single-molecular water hydrogen bond switching adjacent to the ions.

Selective external surface functionalization of large-pore silica materials capable of protein loading

Differentiating the chemical properties of the external and pore surfaces of sol-gel derived mesoporous materials by selective functionalization is important to advancing their application as platforms for biological catalysis, sensing and drug delivery. Prior selective functionalization approaches to concentrating functional groups at the external surface of particles have been limited to small pores (≤5.5 nm diameter) incapable of loading large biological molecules. This work investigates the selective exterior surface functionalization by amines of larger-pored (>7 nm diameter) mesoporous silica particles, which are synthesized by dual surfactant templating and hydrothermal aging. Previously developed selective functionalization techniques rely on choice of functionalization precursor, functionalization reaction time, or pore blocking (by leaving pore templates in as-synthesized materials). The effectiveness of these strategies are compared for larger-pored materials using the precursors (3-aminopropyl)tris(methoxyethoxyethoxy)silane (APTMEES) and (3-aminopropyl)triethoxysilane (APTES). The extent of amine functionalization is determined as a function of precursor reaction time (10 or 20 min) in both as-synthesized and template-extracted materials by confocal laser scanning microscopy of the ∼10 μm diameter particles tagged with fluorescein isothiocyanate. Reaction time, regardless of pore template presence, is demonstrated to be the controlling variable for achieving selective exterior functionalization in these larger pored mesoporous materials. Under the conditions used, 10 min of functionalization with APTMEES localizes amine groups at the exterior of the particles, while 20 min functionalizes both the exterior and the interior pore surfaces. Protein accessibility within pores, before and after selective and full functionalization, is visually confirmed by confocal fluorescence imaging of Rhodamine B tagged lysozyme loaded particles.

Using the Tools and Resources of the RCSB Protein Data Bank.

The Protein Data Bank (PDB) archive is the worldwide repository of experimentally determined three-dimensional structures of large biological molecules found in all three kingdoms of life. Atomic-level structures of these proteins, nucleic acids, and complex assemblies thereof are central to research and education in molecular, cellular, and organismal biology, biochemistry, biophysics, materials science, bioengineering, ecology, and medicine. Several types of information are associated with each PDB archival entry, including atomic coordinates, primary experimental data, polymer sequence(s), and summary metadata. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) serves as the U.S. data center for the PDB, distributing archival data and supporting both simple and complex queries that return results. These data can be freely downloaded, analyzed, and visualized using RCSB PDB tools and resources to gain a deeper understanding of fundamental biological processes, molecular evolution, human health and disease, and drug discovery. © 2016 by John Wiley & Sons, Inc.

Retention of Large Biological Molecules by Size-Exclusion Chromatography

ABSTRACTSize-exclusion chromatography has been developed for the separation of large biological molecules including proteins, polymers, peptides, nucleic acids, and polysaccharide according to their molecular size. This study determined the retention factors for dextran (5, 25, 50, 270, 670, and 1100 kDa) and polysaccharides, such as fucoidan, alginic acid, and laminarin, in the size-exclusion chromatography stationary phase. In addition, the molecular weights and retention factor of three polysaccharides were calculated from the dextran standard curve. The largest retention factor was 4.26 using the size-exclusion chromatography columns (5 kDa dextran). The molecular weights of fucoidan, alginic acid, and laminarin were determined to be 250, 200, and 5 to 64 kDa, respectively.

The kernel energy method (KEM) delivers fast and accurate QTAIM electrostatic charge for atoms in large molecules

The kernel energy method (KEM) is a soft scaling fragment quantum chemical calculation method that proved very useful for the estimation of ab initio energies of very large biological molecules. In this approach, a large molecule is broken into computationally tractable pieces (kernels), which reduces the otherwise rapidly rising computational difficulty with the number of atoms. The formula which delivers the KEM approximation to the total molecular energy includes sums over the energies of double kernels corrected for over-counting by subtracting a sum over single kernels. KEM has been shown to deliver numerically accurate energies of unperturbed large molecules and the energies and dipole moments of large molecules under strong external perturbing electric fields. A recent study has also shown that the full molecular localization–delocalization matrix and the electron density at bond and ring critical point can also be reconstructed from formulae of the same form of the KEM master equation. It is shown that the KEM formula can also be used to reconstruct accurate approximations to the two-particle and one-particle reduced density matrices. This paper shows three numerical examples, of increasing complexity and size, that the atomic charge of an atom in a molecule, calculated from Bader’s quantum theory of atoms in molecules, is accurately predicted from the KEM formula. The error introduced by the KEM approximation is found to be smaller than 0.01 % of the total number of electrons as our calculations demonstrate on three interesting compounds that represent a variety of bonding situations: a highly explosive compound N,N′-dinitrourea (C3H6N4O5, 92 electrons), a strong chelating agent (pyridine-2-azo-p-phenyl)tetramethylguanidine (PAPT) (C16H20N6, 158 electrons), and the conformationally restricted synthetic peptide acetyl-Cα,α-dipropylglycine heptapeptide (Ac-Dpg-7) (C39H71N7O9, 426 electrons).

Theoretical Study of Ultrafast Electron Dynamics in Amino Acids

SynopsisPrompt ionization of large biological molecules may induce ultrafast charge migration along the molecular skeleton, preceding any nuclear rearrangement. This phenomenon has been recently observed in the amino acid phenylalanine in a two-color pump probe experiment, where the production of ionic fragments was measured as a function of the time delay between the two pulses and charge fluctuations manifested as sub-4.5 fs oscillations in the quantum yield of a specific doubly charged fragment. We present our latest results in glycine, and compare with previous findings in phenylalanine. We seek to perform a systematic study including larger aminoacids such as tryptophan.

Trastuzumab and docetaxel in a preclinical organotypic breast cancer model using tissue slices from mammary fat pad: Translational relevance.

With the ever-increasing number of drugs approved to treat cancers, selection of the optimal treatment regimen for an individual patient is challenging. Breast cancer complexity requires novel predictive methods and tools. In the present study, we set up experimental conditions to obtain an 'ex vivo' organotypic culture from xenotransplanted mice aiming at recapitulating the human clinical condition. The effect of trastuzumab (large biological molecule) and docetaxel (small chemical entity) was subsequently investigated on this organotypic model and compared with in vivo and in vitro activity on tumor cells. Tissue slices of 200 µm were obtained from mammary fat pad of SCID mice xenotransplanted with human MCF-7 breast cancer cells. Viability and proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and Ki-67 immunohistochemistry,and apoptosis by cleaved caspase-3 immunohistochemistry. In vivo antitumor activity of trastuzumab and docetaxel was determined by caliper measurement of tumor volume and Ki-67 expression on explanted masses by immunohistochemistry. A Teflon support and normoxia were necessary experimental conditions to obtain high viability of excised breast cancer infiltrated mammary fat pad slices upon 48 h cultivation, as shown by MTT proliferation assay, and Ki-67 expression. Breast cancer tissue slices treated for 48 h with trastuzumab or docetaxel showed a significant dose-dependent reduction of viability by MTT assay. Consistently, both drugs down-modulated Ki-67 and increased cleaved caspase-3. Tumor masses collected from docetaxel-or trastuzumab-treated mice showed a similar reduction of proliferation markers. By contrast, MCF-7 cell cultures were significantly inhibited by docetaxel but not by trastuzumab. Tumor tissue slices represent a more predictive experimental cancer model compared to cell cultures for both small and large molecule antitumor efficacy. This observation supports the relevance of microenvironment in the overall tumor biology and response to therapeutics.

Applicability of the protein environment equilibrium approximation for describing ultrafast biophysical processes

The theoretical description of ultrafast processes in biological systems, in particular, electron transfer in photosynthetic reaction centers, is an important problem in modern biological physics. Because these processes occur in a protein medium with which an energy exchange is possible, methods of the quantum theory of open systems must be used to describe them. But because of a high process rate and the specifics of the protein environment, basic approximations of this theory might be inapplicable. We study the applicability of the approximation of the protein environment (bath) state invariance for the dissipative dynamics of charge transfer between molecule-pigments contained in reaction centers. For this, we use model systems whose parameters are close to real ones. We conclude that this approximation can be used to describe both the monotonic and the oscillating dynamics of the reaction subsystem in large biological molecules. We consider various mechanisms for bath thermalization and show that the bath thermalization occurs not because of the intramolecular redistribution of the vibrational energy in it but because of its coupling to the reaction subsystem.

Electrospray deposition in vacuum as method to create functionally active protein immobilization on polymeric substrates

We demonstrate in this work the deposition of a large biological molecule (fibronectin) on polymeric substrates in a high vacuum environment using an electrospray deposition system. Fibronectin was deposited and its distribution and structure investigated and retention of function (ability to promote cell adhesion) on return to liquid environment is shown. AFM was used to monitor changes in the morphology of the surface before and after fibronectin deposition, whilst the biological activity of the deposited protein is assessed through a quantitative analysis of the biomolecular adhesion and migration of fibroblast cells to the modified surfaces. For the first time we have demonstrated that using high vacuum electrospray deposition it is possible to deposit large protein molecules on polymeric surfaces whilst maintaining the protein activity. The deposition of biological molecules such as proteins with the retention of their activity onto clean well-controlled surfaces under vacuum condition, offers the possibility for future studies utilizing high resolution vacuum based techniques at the atomic and molecular scale providing a greater understanding of protein–surface interface behaviour of relevance to a wide range of applications such as in sensors, diagnostics and tissue engineering.

⁵¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.

Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.

Theoretical study of the complexes of tyrosine and tryptophan with biologically important metal cations in aqueous solutions

In this study geometrical parameters and thermodynamical stability of the complexes formed between zwitterionic tyrosine/tryptophan and some biologically relevant monovalent alkali metal cations (Na+, K+) and divalent alkaline earth metal cations (Mg2+, Ca2+) have been determined on the bases of the new and yet unpublished theoretical calculations performed at the DFT(B3LYP-CAM)/6-31+G(d,p) level in the hydrated environment with the use of the polarizable continuum model (PCM). The obtained results of calculations in aqueous solution indicated that the tyrosine–metal cation and tryptophan–metal cation complexes studied adopted salt bridged (SB) structures involving bidentate coordination of the metal cation (Mn+, n=1, 2) to the carboxylate moiety. In these structures, as inferred from the Mn+⋯O separation range, the metal cation is oriented almost symmetrically to two oxygen atoms of the functional group. It is believed that the theoretical results obtained in this study may be used to gain a better understanding of the interactions between biologically important metal cations and large biological molecules such as proteins.

The Investigation of Zinc-Dependent Enzymes in Pregnant Women and their Correlation to Zinc Deficiency

Zinc is present throughout the body in low concentration, but in most tissues. It performs multiple critical functions and must be supplied at adequate levels consistently or deficiency states will result, from mild to severe. Zinc deficiency is insufficient zinc to meet the needs of biological organisms. Due to its essentiality, a lack of this trace element leads to far more severe and widespread problems. Enzymes are large biological molecules responsible for the thousands of chemical inter-conversions that sustain life. Enzymes are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions. Complementary shape, charge and hydrophilic/ hydrophobic characteristics of enzymes and substrates are responsible for this specificity. Specimens were collected at the University Hospital of Obstetrics and Gynecology “Queen Geraldine” in Tirana, Albania during a period of time from year 2011 to 2013. We took into consideration 500 cases of pregnant women from first to third trimester of pregnancy. Serum zinc was measured by using Photometry (End-Point) method, whereas zinc enzymes such as Alkaline Phosphatase, Amylase, Gamma-glutamyl Transpeptidase, Lactate Dehydrogenase, Creatine Kinase, Aspartate Amino-Transferase, Glutamic Pyruvic Transaminase, Lipase measured with the corresponding methods: Beckman Synchron LX20, Colorimetric ab102523 kit, GenWays GGT kit, nonradioactive colorimetric LDH kit, Max Discovery, Colorimetric kit, Abcam kit and Biovision kit. From 500 cases taken into consideration, 190 pregnant women (38%) had normal zinc values (70 – 120 mcg/dl) 58 pregnant women (11.6%) had zinc levels between 60 – 69.9 mcg/dl, patients which were identified as pregnant women with zinc deficiency, as a consequence of oral contraceptive use; 252 pregnant women (50.4%) with zinc values < 60 mcg/dl, identified as patients with zinc deficiency, a result of malnutrition and also urinary elimination of digestive liquids; 56 pregnant women (11.2%) with zinc values < 30 mcg/dl, identified as patient with definite deficiency as a result of different diseases like acrodermatitis enteropathica ect. According to these results, there was a positive correlation between zinc and enzymes such as ALP, CK and LDH in pregnant women suffering from hypertension, whereas a negative correlation of zinc to enzymes such GGT, AST and GPT in cases with anemia. Zinc prophylactic treatment is important before and during pregnancy. Without a proper nutritional requirement the person falls in the state of zinc deficiency.