The Chromatin Structure of Well-Spread Demembranated Human Sperm Nuclei Revealed by Atomic Force Microscopy

The fundamental structure formed when genomic DNA is packaged by protamine in the human sperm nucleus still remains essentially unresolved. It is known that the binding of protamine, a small arginine-rich protein, to DNA generates a large dense, hydrophobic complex making the sperm chromatin structure difficult to study microscopically. To visualize the internal nuclear structures, isolated human sperm nuclei were swollen extensively in saline buffer using only a reducing agent. The nuclei were swollen during deposition onto coverglass and then imaged in the atomic force microscope (AFM). The two main results obtained from imaging individual well-spread nuclei indicate that native human sperm chromatin is: (1) particulate, consisting primarily of large nodular structures averaging 98 nm in diameter, and (2) also composed of smaller, nucleosome-like particles observed to form linear chains near the nuclear periphery. These two types of chromatin particles imaged by AFM are remarkably similar to other AFM measurements made on native and reconstituted sperm and somatic chromatin

Scanning Tunneling Microscope Images of Adenine and Thymine at Atomic Resolution

The scanning tunneling microscope has been used to obtain images of DNA that reveal its major and minor grooves and the direction of helical coiling, but sufficient resolution has not yet been achieved to identify its bases. To determine if this technology is capable of identifying individual DNA bases, we have examined the molecular arrangements of adenine and thymine attached to the basal plane of highly oriented pyrolytic graphite. Both molecules form highly organized lattices following deposition on heated graphite. Lattice dimensions, structural periodicities, and the epitaxy of adenine and thymine molecules with respect to the basal plane of graphite have been determined. Images of these molecules at atomic resolution reveal that the aromatic regions are strongly detected in both molecules while the various side-groups are not well-resolved. These studies provide the first evidence that tunneling microscopy can be used to discriminate between purines and pyrimidines

Fragmentation of biomolecules using slow highly charged ions

We present first results of biomolecular fragmentation studies with slow highly charged ions (HCI). A layer of the tripeptide RVA was deposited on gold targets and irradiated with slow (few 100 keV) ions, e.g. Xe{sup 50+} and Xe{sup 15+}, extracted from the LLNL EBIT (electron beam ion trap). The secondary ions released upon ion impact were mass analyzed via Time-Of-Flight Secondary-Ion-Mass-Spectrometry (TOF-SIMS). The results show a strong dependence of the positive and negative ion yields on the charge state of the incident ion. We also found that incident ions with high charge states cause the ejection of fragments with a wide mass range as well as the intact molecule (345 amu). The underlying mechanisms are not yet understood but electron depletion of the target due to the high incident charge is likely to cause a variety of fragmentation processes. 6 refs., 2 figs

Application of NMR Methods to Identify Detection Reagents for Use in the Development of Robust Nanosensors

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying bi-molecular interactions at the atomic scale. Our NMR lab is involved in the identification of small molecules, or ligands that bind to target protein receptors, such as tetanus (TeNT) and botulinum (BoNT) neurotoxins, anthrax proteins and HLA-DR10 receptors on non-Hodgkin's lymphoma cancer cells. Once low affinity binders are identified, they can be linked together to produce multidentate synthetic high affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors or biochips for the detection of pathogen proteins in environmental samples or body fluids. Here, we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy (trNOESY) that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known ''marker'' ligand, doxorubicin. Using this assay, we can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site

Electrospray mass spectrometry of NeuAc oligomers associated with the C fragment of the tetanus toxin

The Clostridial neurotoxins, botulinum and tetanus, gain entry into neuronal cells by protein recognition involving cell specific binding sites. The sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides attached to the surface of motor neurons are the suspected recognition and interaction points with Clostridial neurotoxins, although not necessarily the only ones. We have used electrospray ionization mass spectrometry (ESIMS) to examine formation of complexes between the tetanus toxin C fragment, or targeting domain, and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to rapidly and efficiently measure dissociation constants for a number of related NeuAc-containing carbohydrates and NeuAc oligomers, information that has helped identify the structural features of gangliosides that determine their binding to tetanus toxin. The strength of the interactions between the C fragment and (NeuAc){sub n}, are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. The results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer). This study also shows that NeuAc must play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria and viruses to facilitate their entrance into cells

Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously

Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells

Ultra-fast Laser Synthesis of Nanopore Arrays in Silicon for Bio-molecule Separation and Detection

We demonstrate that interference of ultra-fast pulses of laser light can create regular patterns in thin silicon membranes that are compatible with the formation of a uniform array of nanopores. The spacing and size of these pores can be tuned by changing the laser energy, wavelength and number of ultra-short pulses. Short pulses and wavelengths ({approx}550 nm and smaller) are needed to define controllable nanoscale features in silicon. Energy must be localized in time and space to produce the etching, ablation or amorphization effects over the {approx}100 nm length scales appropriate for definition of single pores. Although in this brief study pattern uniformity was limited by laser beam quality, a complementary demonstration reported here used continuous-wave interferometric laser exposure of photoresist to show the promise of the ultra-fast approach for producing uniform pore arrays. The diameters of these interferometrically-defined features are significantly more uniform than the diameters of pores in state-of-the-art polycarbonate track etch membranes widely used for molecular separations

Sexual Selection Halts the Relaxation of Protamine 2 among Rodents

Sexual selection has been proposed as the driving force promoting the rapid evolutionary changes observed in some reproductive genes including protamines. We test this hypothesis in a group of rodents which show marked differences in the intensity of sexual selection. Levels of sperm competition were not associated with the evolutionary rates of protamine 1 but, contrary to expectations, were negatively related to the evolutionary rate of cleaved- and mature-protamine 2. Since both domains were found to be under relaxation, our findings reveal an unforeseen role of sexual selection: to halt the degree of degeneration that proteins within families may experience due to functional redundancy. The degree of relaxation of protamine 2 in this group of rodents is such that in some species it has become dysfunctional and it is not expressed in mature spermatozoa. In contrast, protamine 1 is functionally conserved but shows directed positive selection on specific sites which are functionally relevant such as DNA-anchoring domains and phosphorylation sites. We conclude that in rodents protamine 2 is under relaxation and that sexual selection removes deleterious mutations among species with high levels of sperm competition to maintain the protein functional and the spermatozoa competitive