Laser-Microdissection of Protein Crystals Down to Submicron Dimensions

Abstract

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...

LB Crystallization and Preliminary X-ray Diffraction Analysis of L-Asparaginase from Rhodospirillum rubrum

“…Recently, it was confirmed that proteins can be crystallized in undersaturation conditions using LB nanotemplate [31]. Moreover, the microcrystals obtained by this methods results to be stable to the highly intense synchrotron radiation in comparison with those obtained by classical methods [32].…”

Determination of Protein-Protein Interaction for Cancer Control via Mass Spectrometry and Nanoconductimetry of NAPPA SNAP Arrays: An Overview

“…This, taken together with the unique properties of proteins obtained with Langmuir-Blodgett (LB)-based crystallography [31][32][33][34][35][36][37][38] that can enable new strategies for drug design (work in progress), makes the goal of cancer control tremendously ambitious.…”

Langmuir-Blodgett Nanotemplate Crystallization Combined to Laser-Microfragmentation Uniquely Characterize Proteins Crystals by Synchrotron Microdiffraction

“…Laser-microdissection have recently (Pechkova et al, 2013; been successfully used to dissect Langmuir-Blodgett lysozyme crystals (Nicolini and Pechkova, 2006a;2007;Belmonte et al, 2012) in order to obtain pieces of crystals of very small dimensions in conjuction with X-ray nanodiffraction techniques capable to overcome the very common problem of twinned, defect, aggregated and mosaic crystals.…”

“…Several demonstration experiments show that lasermicrodissection techniques can also be applied to protein crystals (Pechkova et al, 2013;. We have used the term laser-microdissection for the cutting of a crystal by a laser beam into smaller pieces (Pechkova et al, 2013) while microfragmentation is used for the separation of a microdissected crystal into smaller fragments due to effects such as cavitations at domain boundaries and solvent interpenetration.We will explore in this contribution the question whether protein crystals differing in perfection and X-ray radiation stability also differ in microdissection and microfragmentation behavior.…”

“…We have used the term laser-microdissection for the cutting of a crystal by a laser beam into smaller pieces (Pechkova et al, 2013) while microfragmentation is used for the separation of a microdissected crystal into smaller fragments due to effects such as cavitations at domain boundaries and solvent interpenetration.We will explore in this contribution the question whether protein crystals differing in perfection and X-ray radiation stability also differ in microdissection and microfragmentation behavior. Indeed, protein crystals grown by a LangmuirBlodgett (LB) based method (called LB-crystals) are shown here to have a unique higher radiation stability than crystals provided by standard (e.g., hanging drop) crystallization techniques (called standard-crystals) in the wide range of four quite different proteins.…”