Proteins atomic force microscopy

Abstract. Molecular dynamics (MD) simulations of proteins provide descriptions of atomic motions, which allow to relate observable properties of proteins to microscopic processes. Unfortunately, such MD simulations require an enormous amount of computer time and, therefore, are limited to time scales of nanoseconds. We describe first a fast multiple time step structure adapted multipole method (FA-MUSAMM) to speed up the evaluation of the computationally most demanding Coulomb interactions in solvated protein models, secondly an application of this method aiming at a microscopic understanding of single molecule atomic force microscopy experiments, and, thirdly, a new method to predict slow conformational motions at microsecond time scales. 

As an example for an efficient yet quite accurate approximation, in the first part of our contribution we describe a combination of a structure adapted multipole method with a multiple time step scheme (FAMUSAMM — fast multistep structure adapted multipole method) and evaluate its performance. In the second part we present, as a recent application of this method, an MD study of a ligand-receptor unbinding process enforced by single molecule atomic force microscopy. Through comparison of computed unbinding forces with experimental data we evaluate the quality of the simulations. The third part sketches, as a perspective, one way to drastically extend accessible time scales if one restricts oneself to the study of conformational transitions, which arc ubiquitous in proteins and are the elementary steps of many functional conformational motions. 

Whereas STM is best suited for imaging atoms, atomic force microscopy is more appropriate for larger structures. The following image shows a strand of DNA. The two blue regions of the figure are protein molecules bound to the DNA. 

Lee CK, Wang YM, Huang LS, Lin SM. 2007. Atomic force microscopy Determination of unbinding force, off rate and energy barrier for protein-ligand interaction. Micron 38 446-461. 

Polyacrylamide gel electrophoresis results suggest that p-LG undergoes a greater conformational loss as a fimction of extrusion temperature than a-LA, presumably due to intermolecular disulfide bond formation. Atomic force microscopy indicates that texturization results in a loss of secondary structure of aroimd 15%, total loss of globular structure at 78 °C, and conversion to a random coil at 100 °C (Qi and Onwulata, 2011). Moisture has a small effect on whey protein texturization, whereas temperature has the largest effect. Extrusion at or above 75 °C leads to a uniform densely packed polymeric product with no secondary structural elements (mostly a-helix) remaining (Qi and Onwulata, 2011). 

Uricanu, V. I., Duits, M. H. G., and Mellema, J. (2004). Hierarchical networks of casein proteins An elasticity study based on atomic force microscopy. Langmuir 20,5079-5090. 

We studied the surface pressure area isotherms of PS II core complex at different concentrations of NaCl in the subphase (Fig. 2). Addition of NaCl solution greatly enhanced the stability of monolayer of PS II core complex particles at the air-water interface. The n-A curves at subphases of 100 mM and 200 mM NaCl clearly demonstrated that PS II core complexes can be compressed to a relatively high surface pressure (40mN/m), before the monolayer collapses under our experimental conditions. Moreover, the average particle size calculated from tt-A curves using the total amount of protein complex is about 320 nm. This observation agrees well with the particle size directly observed using atomic force microscopy [8], and indicates that nearly all the protein complexes stay at the water surface and form a well-structured monolayer. 

Stine WB Jr, Snyder SW, Ladror US, Wade WS, Miller MF, Perun TJ, Holzman TF, Krafft GA. The nanometer-scale structure of amyloid-beta visualized by atomic force microscopy. J Protein Chem 1996 15 193-203. 

The shift of the amide I mode (FTIR spectra) from 1657 to 1646 cm-1 was attributed to a change in the a-helix native structure to fl-sheets, secondary structure conformations. Atomic Force Microscopy (AFM) images display the coating of the manganese oxide surface as well as the unfolding in a ellipsoidal chain of the protein molecules after adsorption and immobilization on the surface. 

An ordered antibody array has also been assembled on the solid surface by a combination of Langmuir Blodgett (LB) film method and self-assembling method. An ordered monolayer of protein A is deposited on the solid surface by LB method, which is followed by self-assembling of antibody. Individual antigen molecules which are complexed with the antibody array have been quantitated selectively by atomic force microscopy (AFM). 

Bustamante JO, Liepers A, Prendergast RA, Hanover JA, Oberliethner H. Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex. JMembrane Biol 1995 146 X263-X272. 

Chen et al. (2007) have developed a nanoinjector that injects compounds immobilized on MWNT-atomic force microscopy (AFM) tips into the cells. First, a MWNT-AFM tip was fabricated from a normal AFM tip with an MWNT on one end. Next, a compound of interest was immobilized on the MWNT-AFM tip through a disulfide bond linkage. After MWNT-AFM tip was tapped on the cell, the cantilever was further lowered and the MWNT nanoneedle then penetrated the membrane. Once inside the cell, the disulfide linkage was broken under the cells reducing environment and the compound of interest was released inside the cell. The MWNT-AFM tip was then removed from the cell. In this study, protein was... 

Li H, Oberhauser AF, Fowler SB, Clarke J, Fernandez JM. Atomic force microscopy reveals the mechanical design of a modular protein. Proc Natl Acad Sci USA 2000 97 6527-6531. 

Lee, M., et al. (2006) Protein nanoarray on Profinker sirrface constructed by atomic force microscopy dip-pen nanolithography for analysis of protein interaction. Proteomics. 6, 1094-103. 

Ikeda, S., Morris, V.J. (2002). Fine-stranded and particulate aggregates of heat-denatured whey proteins visualized by atomic force microscopy. Biomacromolecules, 3, 382-389. 

Atomic Force Microscopy to Visualize Membrane Proteins... 

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