CRYSTALLIZATION OF BIOLOGICAL MACROMOLECULES MCPHERSON PDF

Protein Structure Analysis pp Cite as. To write about the crystallization conditions for a specific protein or nucleo-protein complex will be of no use to somebody intending to crystallize a completely different molecule or even an homologous one coming from a different organism. Therefore, the emphasis of this chapter is on the general aspects of crystallization and on practical tips concerning the source of the macromolecules, separation techniques, and crystallization conditions. Unable to display preview.

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The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions.

With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality.

Genome-sequencing projects have provided a near complete list of the molecules that are present or potentially present in an organism, and post-genomic projects are aimed at cataloguing the relationships between them.

Understanding metabolic and signaling pathways or gene-regulatory networks relies on a detailed knowledge of protein-metabolite, protein-protein and protein-nucleic acid interactions. X-ray diffraction from high quality crystals remains the most reliable approach to obtain detailed structural information that provides powerful insight into the molecular mechanisms underlying the function of bio-macromolecules and the way they interact to form complex supramolecular assemblies [ 1 ].

Furthermore, knowledge of binding sites at atomic details allows a rational drug design, which is fundamental for searching of new medicines [ 2 ]. For this purpose, protein crystallography is now used at all levels, including target identification and selection [ 3 — 5 ]. The determination of the 3D structure by X-ray crystallography involves essentially six steps: I purification from source or cloning, expression and purification of target macromolecule; II search of initial crystallization conditions; III optimization of crystal quality; IV diffraction data collection; V structure determination and refinement of the 3D model; VI analysis of the refined model.

The advent of high-throughput methods has made the process more efficient [ 6 , 7 ]. Indeed, notable advances in tools for X-ray data collection have been made, including synchrotron beam lines [ 8 ], sensitive X-ray detectors, and improved cryogenic and mounting procedures for crystals.

Once good quality crystals are obtained, the subsequent steps of structure determination can be more safely carried out. However, many times the high-throughput protein structure determination does not give positive results, due to the difficulties of finding conditions that promote growth of high-quality protein crystals. The three stages of crystallization common to all molecules Figure 1 are nucleation, crystal growth and cessation of growth [ 9 — 12 ].

During nucleation an adequate amount of molecules associate in three dimensions to form a thermodynamically stable aggregate, the so called critical nucleus, which provides surfaces suitable for crystal growth. The growth stage, which immediately follows the nucleation, is governed by the diffusion of particles to the surface of the critical nuclei and their ordered assembling onto the growing crystal Figure 1.

Fluid aggregate model. Increasing supersaturation promotes molecule association, which begins to organize into large disordered aggregates a molecules within the cores of such aggregates reorient, redistribute and form more geometrically rigorous interactions; b These latter interactions tend to order and stabilize the aggregate core, which increases to produce a critical nucleus; c This ultimately develops into a true crystal; d Free molecules are then adsorbed to the crystal surface and increase its size by their incorporation into its lattice.

Protein crystal formation requires interactions that are specific, highly directional and organized in a manner that is appropriate for three-dimensional crystal lattice formation. Crystal growth ends when the solution is sufficiently depleted of protein molecules, deformation-induced strain destabilizes the lattice, or the growing crystal faces become poisoned by impurities. The crystallizability of a protein is strictly affected by the chemical and conformational purity and the oligomeric homogeneity of the sample.

The whole crystal growth process can be conveniently visualized in a two-dimensional phase diagram Figure 2 representing the stable states liquid, crystalline, precipitate as a function of two crystallization variables.

When the concentration of a protein solution is brought above its solubility limit, the solution becomes supersaturated. Because these regions are related to kinetic parameters, the boundaries between them are not well defined. The best strategy that should be employed is to induce nucleation at the lowest level of supersaturation just within the labile region.

Following nuclei formation, the concentration of protein in the solution gradually decreases, driving the system into the metastable zone, where growth occurs slowly.

However, it is very difficult to identify these ideal conditions and in order to obtain high-quality crystals it could be necessary to physically separate nucleation and growth steps. Chemical space in crystallization experiments is multidimensional, and several zones may correspond to nucleation and growth of different crystal forms.

It is not yet possible to predict the conditions required to crystallize a protein from its chemico-physical properties. Changes in a single experimental parameter can simultaneously influence several aspects of a crystallization experiment. Despite the importance of protein crystallization, the insights into this process are still limited and currently there are no systematic methods to ensure that ordered three-dimensional crystals will be obtained. This problem has stimulated many efforts to improve the success of protein crystal growth experiments.

The present work will review some of the basic ideas and principles of biological macromolecule crystallization, summarize the standard approaches in crystal growth and illustrate novel tools and strategies to increase the rate of positive results and the diffraction quality of crystals.

Crystal growth of bio-macromolecules is a multi-parametric process as it depends on several factors, such as sample concentration, temperature, pH value, precipitant, buffer, additive, detergent, physical fields, pressure etc. Table 1. Parameters affecting crystallization process [ 10 , 12 , 14 , 15 ]. All these thermodynamic and dynamics variables can be used to control supersaturation in the system and, thus, indirectly, the rates of nucleation and growth.

In the following sections we will describe in details some parameters affecting protein crystallization, which also govern crystallization of other biological macromolecules, such as nucleic acids or macromolecular assemblies.

Chemical and conformational purity of the sample strongly affects the ability to grow crystals. It can be simply assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and mass spectrometry MS.

The light scattered from a solution may be analyzed either in terms of its intensity or in terms of its fluctuations. In the former method, which is called static light scattering, the measure of scattered light intensity as function of angle is used to find the molar mass, the mean squared radius of gyration and the second viral coefficient B The measurements may be also performed at a single angle, provided that the concentration and the refractive index are known.

This procedure has been used to investigate solubility and crystallizability of many macromolecules [ 17 — 20 ]. On the other hand, DLS detects the fluctuations of the scattering intensity due to the Brownian motion of molecules in solution [ 21 , 22 ]. The degree of these fluctuations depends on the diffusion coefficient of the scattering particle, a quantity which is related to the hydrodynamic radius through the Stokes-Einstein equation. The mathematical treatment of the data also permits to assess the degree of homogeneity polydispersity index as well as to construct size distribution.

Furthermore, Saridakis et al. Temperature governs the balance between enthalpy and entropy effects on the free energy. Depending on whether crystallization is enthalpy- or entropy-driven, proteins may become either more or less soluble at higher temperature. Some proteins display a characteristic increased solubility with increasing temperature, whereas others display a decreased, or retrograde solubility [ 24 ]. Furthermore, pKa values of ionizable groups are strictly related to the medium ionic strength.

As a consequence, in the case of proteins with normal solubility, it increases with a temperature increment at low ionic strength, for example if the solution contains components with low dielectric constant, whereas decreases at high ionic strength. In the latter case, however, the solubility variation is very small. The temperature-solubility function is not a property of the protein itself, but is subtly related to the protein-solution system. Equally relevant is the influence of temperature on the rates of nucleation and growth, and on the equilibrium position of the trial.

However, recently many crystallization devices with a fine control of the temperature have been developed to take advantage of the effects of this parameter on the growth mechanism and the crystal form. Proteins generally contain numerous ionizable groups, which have a variety of pKas. As a consequence, protein solubility can dramatically change as pH is altered even by only 0.

The pH affects the detailed nature of protein-protein interactions modifying the possibilities of forming salt bridges and hydrogen bonds crucial to the formation of specific crystal contacts [ 26 ]. Electrostatic interactions, which depend on the protonation state of aminoacid side chains, play a key role in the binding specificity, in protein hydration and in the interactions with small molecules and ions that sometimes mediate the crystal packing contacts.

At a pH characteristic for each protein, called the isoelectric point pI , the positive charges of the molecule exactly balance the negative ones. This would seem to be the best situation for crystal growth as no overall electrostatic repulsion between protein molecules is present. Unfortunately, this idea was not confirmed by an analysis of crystallization conditions of almost ten thousand unique protein crystal forms [ 27 ].

Consequently, a wide pH range has to be explored in the crystallization experiments, but only pH values that maintain the folded structure of the protein are acceptable conditions for protein crystal growth. The correlation between protein thermal stability and probability of yielding crystals is controversial [ 28 ].

However, in many cases pre-crystallization screening based on stability has substantially increased the crystallization success rate [ 29 , 30 ]. This method measures the melting temperature of a protein by monitoring the signal of an external fluorescent probe which interacts with hydrophobic core residues when they become solvent-exposed during the unfolding process [ 31 ]. The low quantity of starting material required for an average thermal shift experiment makes DSF particularly suitable for use in the screening of optimal conditions for protein crystallization targets.

Chemical compounds that reduce protein solubility are referred to as crystallizing or precipitating agents. They reinforce the attractions among bio-macromolecules and act either by altering the activity coefficient of water salts [ 32 ], or by changing the dielectric constant of the solvating medium organic solvents or by increasing molecular crowding high molecular weight polymers like polyethylene glycol, PEG [ 33 ].

Precipitants that act by different mechanisms show little exchangeability: crystals obtained with one type of precipitant do not commonly form if the precipitant is changed with a functionally different one. However, it has been exhaustively demonstrated that combinations of mechanistically distinct precipitating agents can be synergistic and increase the probability of crystal growth [ 34 ] Table 2.

A common approach in crystallization strategies is the use of additives to affect crystal growth and nucleation. Any substance other than the crystallizing compound, the buffer and the precipitant agent is considered as an additive Table 3.

Classification of additives according to McPherson et al. The most common interpretation of specific effects of a given additive on crystal growth is based on the assumption that some specific interactions are established between the additive and particular sites at the protein surface. Very frequently, small salts are used as additive in the crystallization solution. For most proteins the degree of solubility depends weakly on the kind of cation, but strongly on the kind of anion [ 37 ].

In some cases, the ions are essential for the protein biological activity and contribute to maintain certain structural features of the protein. In other cases, metal ions stabilize intermolecular contacts in the crystal.

Recent studies have shown that the application of biocompatible water-soluble ionic liquids, organic salts and salts with melting points at or below room temperature as crystallization additives provides very interesting results [ 39 , 40 ]. Small organic molecules represent another class of very useful additives, which increase long range electrostatic interactions by lowering the dielectric constant, affect solvent structure, and could modify the hydrophobic effect [ 41 — 45 ].

Crystallization of a certain protein often requires the presence in the crystallizing solution of natural ligands, which assure the conformational homogeneity of the sample. These ligands can be chosen on the basis of their binding affinity to the enzyme. Some of these compounds can be added directly to the crystallization drop co-crystallization , others need to be bound to the protein in advance.

Since the pioneering studies on additive effects on protein crystal growth [ 36 , 46 , 47 ], the use of these molecules has become extremely widespread. In the literature many papers discuss the action and the effects of these substances on the protein crystallizability and very recently McPherson et al. In this technique, called the cross-influence procedure CIP , each crystallization trial utilizes four droplets containing equal volume of the precipitating agent.

The protein is added to one of the droplets whereas additives metal salts are placed in the others, then all drops are left to equilibrate against the same reservoir. The presence of the drops without sample should slightly change the vapor pressure of water over the drop containing the protein. Furthermore, when the precipitant solution contains volatile buffers, the pH of the drop with the protein may slightly change, due to vapor diffusion of the volatile acid or base component.

During the crystal growth, the solution around the crystal gets depleted in protein and becomes less dense than the bulk solution. Therefore a density gradient is created that in conjunction with the gravitational field leads to buoyancy driven convection close to the crystal [ 50 ].

Furthermore, the growing crystal surfaces come in contact with bulk solution that is typically several times supersaturated. These effects are harmful, because they interfere with the correct addition of protein molecules to the growing crystal lattice and may cause crystal disorder. In zero gravity no buoyancy-driven convection occurs and the growing crystal does not move with respect to the surrounding fluid.

The matter is transported in a purely diffusive way [ 51 ] and the crystal growth takes place under ideal conditions where the growing surface is in contact with a solution that is slightly supersaturated.

SHRI SAINATH BHAJAN MALA PDF

An Overview of Biological Macromolecule Crystallization

For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for X-ray data collection. Even when the initial samples are suitable, often marginally, refinement of conditions is recommended in order to obtain the highest quality crystals that can be grown. The quality of an X-ray structure determination is directly correlated with the size and the perfection of the crystalline samples; thus, refinement of conditions should always be a primary component of crystal growth.

GENU FLEXUM PDF

Crystallization of Biological Macromolecules

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