Atomic force microscopy of protein films and crystals

Pechkova, Eugenia; Sartore, Marco; Giacomelli, Luca; Nicolini, Claudio

doi:10.1063/1.2785032

Cited by 17 publications

(16 citation statements)

References 19 publications

(24 reference statements)

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“…As shown previously [14] and confirmed here by ID11 GISAXS evaluation the in plane structure distance is indeed decreasing with heat compatibly with the merging of layers macromolecular structures, with the decrease in the plane length being likely due to the loss of water caused by LB formation and heating to 150°C. The comparative atomic structure characterization of thermophilic versus mesophilic proteins by X-ray crystallographic diffraction and nanogravimetric analysis [19] in protein solution, thin film and crystal has recently allowed to draw a coherent explanation for the origin and the molecular…”

Section: Discussionmentioning

confidence: 99%

“…Images of the protein thin LS films have been obtained in tapping mode with a cantilever I type NSC14/Cr-Au MikroMash in a dry atmosphere utilizing an instrument based on the SPMagic controller built in house [7,19]. The freeware WSxM (http://www.nanotec.es) was utilized for the processing of the acquired images.…”

Section: Synchrotron Diffraction Of Multilayered Ls Pga Films After Hmentioning

confidence: 99%

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Synchrotron Diffraction of Multilayered LS PGA Films after Heating and Cooling

Nicolini¹,

John²,

Pechkova³

2015

NanoWorld J

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X-ray diffraction patterns of multilayered Langmuir-Schaefer (LS) film of penicillin G acylase (PGA) enzyme were acquired at the ID11 of Synchrotron Radiation at ESRF (Grenoble, France). In addition to what shown by GISAXS at ID13 and by AFM, the ID11 beamline appears capable to monitor the diffraction and structural properties of the Langmuir-Shaefer multilayered PGA enzyme film similar to what apparent in the corresponding PGA crystals. The dramatic increase of long-range order in the LB multi-layered enzyme films after heating and cooling, made previously apparent by grazing incidence small angle X-ray scattering using ID13 microbeam, was here utilized at the ID11 beamline to yield unique diffraction patterns of the PGA LB linked to the enzyme atomic structure. This could open the way to bypass the bottleneck of protein crystallization which is leaving still unsolved large part of important proteins, like the membrane ones.

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Section: Discussionmentioning

confidence: 99%

Section: Synchrotron Diffraction Of Multilayered Ls Pga Films After Hmentioning

confidence: 99%

Synchrotron Diffraction of Multilayered LS PGA Films after Heating and Cooling

Nicolini¹,

John²,

Pechkova³

2015

NanoWorld J

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“…(Figures 3A-Figure 3C) Indeed, Figure 3A shows a standard-crystal, 20 minutes after only two laser pulses focused on one point. The morphology revealed displays large differences between standard-and LB-crystals [26,43,44]. Images of microdissected and microfragmentated lysozyme LB-crystals in a cryoloop are shown in Figures 4A-4C.…”

Section: Laser-microdissectionmentioning

confidence: 99%

“…Lysozyme protein crystals were obtained by a state-of-theart crystallization technique based on Langmuir-Blodgett (LB) nanotemplates [28,[40][41][42][43][44][45][46][47]. LB-nanofilms were generated by the LBtechnique and its variation, a modified Langmuir-Schaeffer (LS) technique [27,28,42].…”

Section: Protein Crystallizationmentioning

confidence: 99%

Laser-Microdissection of Protein Crystals Down to Submicron Dimensions

Pechkova¹

2013

J Nanomed Nanotechnol

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IntroductionWhile, a lot of efforts are being invested at Synchrotron Radiation (SR) sources on high-throughput protein-to-structure pipe-lines in the context of structural genomics [1][2][3][4], methods allowing manipulating and tailoring of protein micro-crystals in a laboratory or SR-beamline environment have been less explored. Such techniques could, however, be useful for separating and sorting crystals from a batch, for dissection of crystals, polymorphs or crystalline domains from an aggregate or a cellular environment. We also note the idealized model of a protein crystal composed of coherently scattering mosaic blocks [5] which can be experimentally studied by X-ray line-profile analysis or X-ray topography [6,7]. The properties and interactions of individual mosaic blocks are, however, rarely addressed. As an example we mention the observation of radiation damage induced accumulation of hydrogen at the mosaic-block boundaries of insulin crystals resulting in a reduction of block-size and increase of mosaicity [8,9]. A more detailed understanding of mosaic block interactions could contribute to more refined models of protein crystal mechanics and is of fundamental importance for defining the frontiers of nanomedicine [10,11]. Finally we note that raster-diffraction techniques allow identifying highquality diffracting volume-elements in a batch of microcrystals or a larger crystal [12,13]. We are aware that the selection of mosaic blocks for X-ray nanodiffraction techniques would represent a methodological progress and could in particular improve the quality of structural refinements for high mosaicity crystals. For all of these reasons it is of interest exploring techniques allowing dissecting protein crystals, ultimately down to the level of individual mosaic blocks.In view of the generally fragile nature of biological matter, laser-optical techniques appear to be well suited for protein crystal manipulation [9,[14][15][16] and microdissection [17][18][19][20][21][22][23][24][25]. Our aim was exploring the ultimate size in coherently scattering crystal fragments obtainable by microdissection, principally for the model protein lysozyme. Preliminary results have been reported elsewhere in Pechkova and Nicolini [26]. In the following, we will use the term laser-microdissection for the cutting of a crystal by a laser beam while microfragmentation will be used for the separation of a microdissected crystal into smaller fragments due to effects such as cavitations at domain boundaries and solvent interpenetration. Given the rather small microfragments obtained in the present study, we used X-ray nanocrystallography for localizing individual microfragments and collecting data sets. We were also interested in the question whether crystals differing in perfection and X-ray radiation stability differ also in microdissection and microfragmentation behavior. This issue is of importance for the generation of very small crystals which might be more prone to laser-radiation damage than larger crystals. We will compar...

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“…The growth of protein (such as, for example, Cytochrome P450scc, Thaumatin and Lysozyme) films on homologous crystal surfaces has been studied and characterized by Atomic Force Microscopy (AFM) [26][27][28], but microscopic details of the crystal growth process are still poorly known.…”

Section: Introductionmentioning

confidence: 99%