New Methods for Depositing and Imaging Molecules in Scanning Tunneling Microscopy

Methods and apparatus are described to deposit and image molecules by scanning tunneling microscopy (STM) under an inert atmosphere. Three methods of applying molecules have been evaluated: equilibrium adsorption from the vapor phase, sublimation, and electrospraying. Using these methods, a variety of organic and biopolymer molecules have been deposited and imaged on graphite and on gold (111), grown epitaxially on mica. Compared with alternatives, such as the use of high vacuum apparatus or glove boxes, these procedures offer some important advantages: they are inexpensive, convenient, and more rapid. Mercaptoethanol, ethanolamine, ethanol, acetic acid, and water produce two-dimensional crystalline adlayers on gold substrates, when they are introduced into the scanning cell as vapors. These adlayers are assumed to involve hydrogen bonding of the molecules to an oxide of gold formed on the surface. Electrospraying protein solutions on gold surfaces yielded images of individual protein molecules with lateral dimensions close to those measured by X-ray analysis, and thicknesses of 0.6-1.3 nm. In the case of metallothionein, the known internal domain structure of the molecule was reproducibly observed. No detailed internal structure could be resolved in the other examples examined

Effects Of Unpaired Bases On The Conformation And Stability Of 3-arm Dna Junctions

Three-arm DNA junctions, in which three double helices intersect at a branch, have unique structure and reactivity of bases at and near the branch. Their solution conformation is asymmetric in the presence of Mg2+, while bases at the branch are sensitive to single-strand-specific agents. Following the surprising report that unpaired bases at the branch stabilize three-arm junctions, we have investigated the geometry and thermodynamics of three-arm junctions containing pendant T and A bases. The results are consistent with additional structure formation in junctions containing up to four pendant bases at the branch: relative to the tight junction, the thermal stability of junctions with two T\u27s or A\u27s at the branch increases; bases near the branch become less reactive to single-strand-reactive probes; and the enthalpy of formation is more negative. The interaction of ethidium observed at the branch in three-arm junctions is enhanced in junctions with unpaired bases at the branch. The geometry of three-arm junctions is perturbed by the presence of pendant bases, as seen by measuring the electrophoretic mobility of junctions to which long duplex arms are appended pairwise

Helix formation and the unfolded state of a 52-residue helical protein

A growing class of proteins in biological processes has been found to be unfolded on isolation under normal solution conditions. We have used NMR spectroscopy to characterize the structural and dynamic properties of the unfolded and partially folded states of a 52-residue alanine-rich protein (Ala-14) at temperatures from −5°C to 40°C. At 40°C, alanine residues in Ala-14 adopt φ and ψ angles, consistent with a significant ensemble population of polyproline II conformation. Analysis of relaxation rates in the protein reveals that a series of residues, Gln 35–Ala 36–Ala 37–Lys 38–Asp 39–Asp 40–Ala 41–Ala 42, displays slow motional dynamics at both −5°C and 40°C. Temperature-dependent chemical shift changes indicate that this region is the site of helix initiation. The remaining N-terminal residues become increasingly dynamic as they extend from the nucleation site. The C terminus remains dynamic and changes less with temperature, indicating it is relatively unstructured. Ala-14 provides a high-resolution portrait of the unfolded state and the process of helix nucleation and propagation in the absence of tertiary contacts, information that bears on early events in protein folding