The BPTI decamer observed in acidic pH crystal forms pre-exists as a stable species in solution

Hamiaux, Cyril; Pérez, Javier; Prangé, Thierry; Veesler, Stéphane; Riès-Kautt, Madeleine; Vachette, Patrice

doi:10.1006/jmbi.2000.3584

Cited by 60 publications

(79 citation statements)

References 54 publications

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“…For a nonaggregating system, q m increases with c and the low angle peak in S M (q) moves to higher q values. SAXS and SANS are widely used to study interactions in protein solutions, e.g., at the onset of crystallization (18)(19)(20)(21).…”

Section: ]mentioning

confidence: 99%

Absence of equilibrium cluster phase in concentrated lysozyme solutions

Shukla

Mylonas

Cola

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

177

202

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In colloidal systems, the interplay between the short range attraction and long-range repulsion can lead to a low density associated state consisting of clusters of individual particles. Recently, such an equilibrium cluster phase was also reported for concentrated solutions of lysozyme at low ionic strength and close to the physiological pH. Stradner et al. [(2004) Equilibrium cluster formation in concentrated protein solutions and colloids. Nature 432:492-495] found that the position of the low-angle interference peak in small-angle x-ray and neutron scattering (SAXS and SANS) patterns from lysozyme solutions was essentially independent of the protein concentration and attributed these unexpected results to the presence of equilibrium clusters. This work prompted a series of experimental and theoretical investigations, but also revealed some inconsistencies. We have repeated these experiments following the protein preparation protocols of Stradner et al. using several batches of lysozyme and exploring a broad range of concentrations, temperature and other conditions. Our measurements were done in multiple experimental sessions at three different high-resolution SAXS and SANS instruments. The lowionic-strength lysozyme solutions displayed a clear shift in peak positions with concentration, incompatible with the presence of the cluster phase but consistent with the system of repulsively interacting individual lysozyme molecules. Within the decoupling approximation, the experimental data can be fitted using an effective interparticle interaction potential involving short-range attraction and long-range repulsion. dynamic arrested state ͉ macromolecular solutions ͉ protein interactions ͉ small-angle scattering ͉ structure factor T he arrested dynamics of colloidal systems and protein solutions interacting via short-range interactions have been actively studied both theoretically and experimentally in recent years (1-4). The mode coupling theory and molecular dynamics (MD) simulations have successfully unified seemingly dissimilar dynamical arrest scenarios in colloidal systems (4, 5). In addition to the conventional glassy state induced by the packing constraints, the presence of short-range attraction leads to a different glassy behavior. The apparently diverse type of dynamical arrest, such as gelation, jamming, glassification or non-ergodicity transition, etc., found in attractive systems can be unified in terms of this attractive glass transition (3). As competing shortrange attraction and long-range repulsion are introduced, additional features are observed (5). In particular, at intermediate volume fractions, the colloidal particles can form an equilibrium cluster phase, which in turn stabilizes a low-density arrested state (6). This type of particle clustering process at low volume fractions has been observed for various colloidal systems (7-9).Although the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory successfully describes the microstructure and equilibrium phase behavior of charged colloidal systems ov...

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Section: ]mentioning

confidence: 99%

Absence of equilibrium cluster phase in concentrated lysozyme solutions

Shukla

Mylonas

Cola

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

177

202

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“…33 Calculated form factors for BPTI monomer and decamer were obtained using the CRYSOL program 55 from a set of crystallographic coordinates (PDB entry 1BHC 34 ). At very low scattering vectors, (s < 0.02 Å -1 ), interparticle interactions may perturb the scattering intensity and affect the form factor.…”

Section: Simulation Procedures 4321 Form Factorsmentioning

confidence: 99%

Exploring Bovine Pancreatic Trypsin Inhibitor Phase Transitions

et al. 2006

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This paper presents an investigation of the phase diagram of BPTI (bovine pancreatic trypsin inhibitor)/350 mM KSCN at pH 4.9 by direct observation and numerical simulations. We report optical microscopy and light and X-ray scattering experiments coupled with theoretical data analysis using numerical tools. The phase diagram is thoroughly determined, as a function of temperature. Two polymorphs are observed by video microscopy and their solubility measured. In this phase diagram, the liquid-liquid phase separation (LLPS) is metastable with respect to the solid-liquid phase separation. Above the T L-L boundary curve, solutions are composed of a mixture of BPTI monomers and decamers. Attractive interactions are stronger between decamers than between monomers. Below the T L-L boundary curve, the dense phase is highly concentrated in protein and composed of BPTI decamers alone. Thus, the driving force for liquid-liquid or liquid-solid phase separation is the attraction between decamers at low pH. The structure factors of the dense phases are characteristic of repulsive dense phases because of a hard sphere repulsion core, meaning that in the dense phase proteins are actually in contact (interparticle distance of 53 Å). In agreement with the Oswald rule of stages, LLPS occurs prior to and impedes the solid nucleation.

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“…SAXS can thus be used as a method for the rapid screening of samples in various aqueous solvents/ additives, including e.g. identification and optimization of crystallization conditions (Bonnete et al, 1999;Hamiaux et al, 2000).…”

Section: Introductionmentioning

confidence: 99%