Use of dynamic light scattering to assess crystallizability of macromolecules and macromolecular assemblies.

DLS has been used to develop models of crystal nucleation and growth and to monitor the formation of nuclei versus amorphous aggregates as a function of solvent conditions and protein concentration. Early light scattering studies of lysozyme provided experimental support for a model in which crystallization is a process involving co-operative, step-wise addition of identical molecules to a growing ordered assembly, whereas precipitation results from non-specific aggregation of molecules into a heterogeneous polymer [7xOn the crystallization of proteins. Kam, Z., Shore, H.B., and Feher, G. J. Mol. Biol. 1978; 123: 539–555Crossref | PubMed | Scopus (212)See all References[7]. With modern DLS instrumentation, Wilson [8xMonitoring crystallization experiments using dynamic light scattering: assaying and monitoring protein crystallization in solution. Wilson, W.W. Methods. 1990; 1: 110–117Crossref | Scopus (30)See all References[8] distinguished solution conditions yielding crystals or amorphous aggregates and studied the thermodynamic properties of these two distinct processes. DLS studies of model proteins approaching supersaturation have also demonstrated that precipitants causing aggregation prior to saturation failed to produce crystals, while precipitants with which the protein remained monodisperse up to the point of nucleation gave crystals [9xDiagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering. Mikol, V., Hirsch, E., and Giege, R. J. Mol. Biol. 1990; 213: 187–195Crossref | PubMed | Scopus (123)See all References[9].These observations suggest that ‘well behaved’, mono-disperse preparations of macromolecules or macromolecular assemblies can be crystallized, whereas samples that aggregate randomly cannot. Zulauf and D'Arcy [10xLight scattering of proteins as a criterion for crystallization. Zulauf, M. and D'Arcy, A. J. Cryst. Growth. 1992; 122: 102–106Crossref | Scopus (89)See all References[10] tested this hypothesis using DLS to analyze the aggregation state of 15 proteins, which had already been subjected to extensive crystallization trials. They obtained a 100 % correlation between monodispersity and crystallizability (9/9 cases) and a corresponding strong correlation between the presence of aggregates under typical solution conditions (0.1– 0.5 mg ml −1 in appropriately buffered solutions) and failure of crystallization trials (5/6 cases).Figure 1Distribution of apparent molecular weight determined by DLS for seven protein–DNA complexes. Success achieved in crystallizing each complex is indicated. (a) Complete USF protein and the carboxy-terminal truncated protein USF(1–260). (b) USF protein with amino-terminal deletion [USF(196–310)] and with a further carboxy-terminal deletion [USF(196–260)]. (c) Carboxy-terminal deletion of Max(1–113) and two further amino- terminal deletions [Max (11–113) and Max(22–113)]. All proteins were mixed with stoichiometric amounts of duplex oligonucleotide with the appropriate binding site. The measurements were performed using a DynaPro-801 Molecular Sizing Instrument (Protein Solutions, Inc., Charlottesville, VA) with the protein– DNA complexes at concentrations of 1–3 mg ml −1 in 100 mM KCl, 10 mM Hepes-KOH pH 7.5, (and 5 mM dithiothreitol for proteins with cysteines). These data underscore the difference between DLS and gel filtration, which cannot distinguish between broad unimodal and narrow unimodal size distributions that do crystallize.View Large Image | View Hi-Res Image | Download PowerPoint SlideThis remarkable correlation between monodispersity and crystallizability was also apparent during our crystallographic studies of helix-loop-helix (HLH) transcription factors complexed with their specific DNA target sequences. Upstream Stimulatory Factor (USF), a 34 kDa HLH protein originally purified from HeLa nuclear extract [11xThe adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer. Gregor, P.D., Sawadogo, M., and Roeder, R.G. Genes Dev. 1990; 4: 1730–1740Crossref | PubMedSee all References[11], was overexpressed in Escherichia coli, purified to apparent homogeneity, and shown to be fully active in DNA binding [12xStructure and function of the b/HLH/Z domain of USF. Ferre-D'Amare, A.R., Pognonec, P., Roeder, R.G., and Burley, S.K. EMBO J. 1994; 13: 180–189PubMedSee all References[12]. Despite very considerable efforts, no crystals of either the USF apo-protein or USF–DNA complexes were obtained. Inspection of the aggregation state of the 34 kDa protein by DLS immediately revealed severe aggregation (Figure 1aFigure 1a). The distribution of apparent molecular weight is very broad, irregular, and extends to well beyond two million daltons. A carboxy-terminal truncation of the protein [USF(1–260)] did not improve the biochemical behavior of the protein. However, elimination of the amino-terminal activation domain did result in a dramatic reduction in aggregation (Figure 1bFigure 1b). USF(196–310) contains an intact, fully active, DNA-binding domain plus some additional carboxy-terminal residues, and its complex with DNA demonstrates a broad unimodal distribution of apparent molecular weight when measured by DLS, with no evidence of high-order aggregation. A further carboxy-terminal truncation to yield the minimal DNA-binding polypeptide [USF(196–260)] resulted in a protein–DNA complex that exhibited a narrow unimodal distribution of apparent molecular weight and readily crystallized upon addition of precipitants. Optimization of the crystallization conditions by conventional approaches yielded diffraction-quality co-crystals and rapid determination of a 2.9 a resolution X-ray structure followed [12xStructure and function of the b/HLH/Z domain of USF. Ferre-D'Amare, A.R., Pognonec, P., Roeder, R.G., and Burley, S.K. EMBO J. 1994; 13: 180–189PubMedSee all References[12].A similar construct-screening strategy was applied to Max [[13]xAssociation of Myn, the murine homolog of Max with c-Myc stimulates methylation-sensitive DNA binding and Ras cotransformation. Prendergast, G.C., Lawe, D., and Ziff, E.B. Cell. 1991; 65: 395–407Abstract | Full Text PDF | PubMed | Scopus (332)See all References, [14]xMax: a helix- loop-helix zipper protein that forms a sequence specific DNA- binding complex with Myc. Blackwood, E.M. and Eisenman, R.N. Science. 1991; 251: 1211–1217Crossref | PubMedSee all References], another member of the HLH family of transcription factors (Figure 1cFigure 1c). The Max(1–113)–DNA complex exhibited a broad unimodal distribution of apparent molecular weight when inspected by DLS. Although some crystalline precipitates were obtained during standard crystallization trials, single crystals were not forthcoming from this construct. Elimination of 10 amino-terminal residues to yield Max(11–113) aggravated the problem, producing a highly aggregated protein. In contrast, deletion of 11 more residues produced a ‘well-behaved’ protein [Max(22–113)], which was still fully active in DNA-binding assays. DLS studies of the Max(22–113)–DNA complex demonstrated that the preparation was monodisperse with a narrow, unimodal distribution of apparent molecular weight. When the material used for DLS was recovered from the sample cell, concentrated, and precipitants added, single crystals were obtained immediately and a 2.9 a resolution X-ray structure of the complex has been reported [15xRecognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Ferre-D'Amare, A.R., Prendergast, G.C., Ziff, E.B., and Burley, S.K. Nature. 1993; 363: 38–45Crossref | PubMedSee all References[15].Further examples of the correlation between monodispersity and crystallizability have been obtained with a number of different macromolecular systems, both in our laboratory and elsewhere. The DLS experiment is quick (a few minutes), non-destructive and requires a minimum of purified material (typically < 1mg). Macromolecular samples can be assayed routinely for monodispersity as a function of solvent conditions, or the presence of ligands, inhibitors or cofactors. Similarly, monodispersity can be measured following post-translational modifications or partial proteolysis, etc. We believe that such measurements greatly enhance the likelihood of successful crystallization trials. Sample monodispersity is also critical for other biophysical methods, including nuclear magnetic resonance spectroscopy and small-angle X-ray and neutron scattering. DLS should be a useful screening step for samples prepared for these solution-based techniques. Finally, DLS may represent an important method for quality control during manufacture of recombinant proteins for therapeutic use.