Alexander J. Malkin

Imaging of viruses by atomic force microscopy

Atomic force microscopy (AFM) has been used to image a variety of virus particles in vitro and could, conceivably, be used as a useful diagnostic for their presence, their structural characterization and even their identification. Virus particles can be imaged by AFM in air, under alcohol or in physiological medium, and accurate measurements of their dimensions obtained. In addition, the appearance and organization of capsomere structures on their surfaces are frequently visible. A number of viruses and virus crystals have been imaged successfully using AFM and improvements in AFM technology and sample handling will undoubtedly increase even more its power, resolution and scope.

The effects of microgravity on protein crystallization: evidence for concentration gradients around growing crystals

Abstract Atomic force microscopy (AFM) investigations have revealed that macromolecular crystals, during their growth, incorporate an extensive array of impurities. These vary from individual molecules to large particles, and microcrystals in the micron size range. AFM, along with X-ray topology, has further shown that the density of defects and faults in most macromolecular crystals is very high in comparison with conventional crystals. The high defect density is a consequence of the incorporation of impurities, misoriented nutrient molecules, and aggregates of molecules. High defect and impurity density, contributes to a deterioration of both the mechanical and the diffraction properties of crystals. In microgravity, access by impurities and aggregates to growing crystal surfaces is restricted due to altered fluid transport properties. We designed, and have now constructed an instrument, the observable protein crystal growth apparatus (OPCGA) that employs a fused optics, phase shift, Mach–Zehnder interferometer to analyze the fluid environment around growing crystals. Using this device, which will ultimately be employed on the international space station, we have, in thin cells on earth, succeeded in directly visualizing concentration gradients around growing protein crystals. This provides the first direct evidence that quasi-stable depletion zones formed around growing crystals in space may explain the improved quality of macromolecular crystals grown in microgravity. Further application of the interferometric technique will allow us to quantitatively describe the shapes, extent, and magnitudes of the concentration gradients and to evaluate their degree of stability.

AFM studies of the nucleation and growth mechanisms of macromolecular crystals

Abstract Atomic force microscopy (AFM) has been used to visualize events arising from the formation of intervening metastable phases at the surfaces of macromolecular crystals growing from solution. Crystals investigated were of the proteins canavalin, thaumatin, lipase, xylanase, and catalase, crystals of transfer RNA, and crystals of satellite tobacco mosaic virus. The appearance of aggregates on crystal surfaces was observed. The aggregates we infer to originate from liquid-protein droplets. These were particularly evident in freshly mixed mother liquor solutions. Droplets, upon sedimentation, have two possible fates. In some cases they immediately restructured as crystalline, multilayer stacks whose development was guided by, and contiguous with the underlying lattice. These contributed to the ordered growth of the crystal by serving as sources of growth steps. In other cases, liquid-protein droplets formed distinct microcrystals, somehow discontinuous with the underlying lattice, and these were subsequently incorporated into the growing substrate crystal. Scarring experiments with the AFM tip indicated that, detached from the crystal, molecules do not dissolve in the fluid phase but form metastable liquid-protein droplets with a potential to rapidly crystallize on the crystal surface. The molecular structure of the growth steps for thaumatin and lipase protein crystals were deduced. There is no step roughness due to thermal fluctuations, and each protein molecule which incorporated into the step edge remained. Growth steps propagate by addition of individual molecules which form subkinks of different size on the step edge.

Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers

Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better with dodecylamine but not with a 1:1 chelate of Ca(2+) and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results provide new insight into the role of the coat layer in spore properties.

Rapid visualization at high resolution of pathogens by atomic force microscopy: Structural studies of herpes simplex virus-1

A relatively crude preparation of herpes simplex virus was rapidly visualized by atomic force microscopy after exposure to conditions that produced gradual degradation of the virions. Images were obtained of 1) the intact, enveloped virus; 2) the underlying capsid with associated tegument proteins along with fragments of the membrane; 3) the capsomeres composing the capsid and their surface arrangement; 4) damaged and partially degraded capsids with missing capsomeres; and 5) the DNA extruded from damaged virions. These images provide a unique perspective on the structures of individual virus particles. Atomic force microscopy can thus be used as a diagnostic tool to provide a rapid way to obtain high-resolution images of human pathogens from crude preparations. It is a useful technique that complements X-ray-based structure determination, cryo-electron microscopy techniques, and optical microscopies in the field of molecular pathogenesis.

Macromolecular crystal growth as revealed by atomic force microscopy

Direct visualization of macromolecular crystal growth using atomic force microscopy (AFM) has provided a powerful tool in the delineation of mechanisms and the kinetics of the growth process. It has further allowed us to evaluate the wide variety of impurities that are incorporated into crystals of proteins, nucleic acids, and viruses. We can, using AFM, image the defects and imperfections that afflict these crystals, the impurity layers that poison their surfaces, and the consequences of various factors on morphological development. All of these can be recorded under normal growth conditions, in native mother liquors, over time intervals ranging from minutes to days, and at the molecular level.

The liquid protein phase in crystallization: a case study—intact immunoglobulins

Abstract A common observation by protein chemists has been the appearance, for many proteins in aqueous solutions, of oil like droplets, or in more extreme cases the formation of a second oil like phase. These may accompany the formation of precipitate in “salting out” or “salting in’ procedures, but more commonly appear in place of any precipitate. Such phase separations also occur, with even greater frequency, in the presence of polymeric precipitants such as polyethyleneglycol (PEG). In general the appearance of a second liquid phase has been taken as indicative of protein aggregation, though an aggregate state distinctly different from that characteristic of amorphous precipitate. While the latter is thought to be composed of linear and branched assemblies, polymers of a sort, the oil phase suggests a more compact, three-dimensional, but fluid state. An important property of an alternate, fluid phase is that it can mediate transitions between other states, for example, between protein molecules free in solution and protein molecules immobilized in amorphous precipitate or crystals. The “liquid protein” phase can be readily observed in many crystallization experiments either prior to the appearance of visible crystals, or directly participating in the crystal growth process. In some cases the relationship between the liquid phase and developing crystals is intimate. Crystals grow directly from the liquid phase, or appear only after the visible formation of the liquid phase. We describe here our experience with a class of macromolecules, immunoglobulins, and particularly IDEC-151, an IgG specific for CD4 on human lymphocytes. This protein has been crystallized from a Jeffamine-LiSO 4 mother liquor and, its crystallization illustrates many of the features associated with the liquid protein, or protein rich phase.

Imaging Cell Wall Architecture in Single Zinnia elegans Tracheary Elements

The chemical and structural organization of the plant cell wall was examined in Zinnia elegans tracheary elements (TEs), which specialize by developing prominent secondary wall thickenings underlying the primary wall during xylogenesis in vitro. Three imaging platforms were used in conjunction with chemical extraction of wall components to investigate the composition and structure of single Zinnia TEs. Using fluorescence microscopy with a green fluorescent protein-tagged Clostridium thermocellum family 3 carbohydrate-binding module specific for crystalline cellulose, we found that cellulose accessibility and binding in TEs increased significantly following an acidified chlorite treatment. Examination of chemical composition by synchrotron radiation-based Fourier-transform infrared spectromicroscopy indicated a loss of lignin and a modest loss of other polysaccharides in treated TEs. Atomic force microscopy was used to extensively characterize the topography of cell wall surfaces in TEs, revealing an outer granular matrix covering the underlying meshwork of cellulose fibrils. The internal organization of TEs was determined using secondary wall fragments generated by sonication. Atomic force microscopy revealed that the resulting rings, spirals, and reticulate structures were composed of fibrils arranged in parallel. Based on these combined results, we generated an architectural model of Zinnia TEs composed of three layers: an outermost granular layer, a middle primary wall composed of a meshwork of cellulose fibrils, and inner secondary wall thickenings containing parallel cellulose fibrils. In addition to insights in plant biology, studies using Zinnia TEs could prove especially productive in assessing cell wall responses to enzymatic and microbial degradation, thus aiding current efforts in lignocellulosic biofuel production.

Atomic force microscopy applications in macromolecular crystallography

Atomic force microscopy (AFM) can be applied both in situ and ex situ to study the growth of crystals from solution. The method is particularly useful for investigating the crystallization of proteins, nucleic acids and viruses because it can be carried out in the mother liquor and in a non-perturbing fashion. Interactions and transformations between various growth mechanisms can be directly visualized as a function of supersaturation, as can the incorporation of diverse impurities and the formation and propagation of defects. Because the crystals can be observed over long periods, it is also possible to obtain precise quantitative measures of the kinetic parameters for nucleation and growth. Finally, AFM has allowed us to identify a number of previously unsuspected phenomena that influence nucleation, rate of growth and the ultimate perfection of macromolecular crystals. These are all features which are important in determining the ultimate resolution and quality of a crystals diffraction pattern.

Spore Coat Architecture of Clostridium novyi NT Spores

Spores of the anaerobic bacterium Clostridium novyi NT are able to germinate in and destroy hypoxic regions of tumors in experimental animals. Future progress in this area will benefit from a better understanding of the germination and outgrowth processes that are essential for the tumorilytic properties of these spores. Toward this end, we have used both transmission electron microscopy and atomic force microscopy to determine the structure of both dormant and germinating spores. We found that the spores are surrounded by an amorphous layer intertwined with honeycomb parasporal layers. Moreover, the spore coat layers had apparently self-assembled, and this assembly was likely to be governed by crystal growth principles. During germination and outgrowth, the honeycomb layers, as well as the underlying spore coat and undercoat layers, sequentially dissolved until the vegetative cell was released. In addition to their implications for understanding the biology of C. novyi NT, these studies document the presence of proteinaceous growth spirals in a biological organism.