In situ study of the state of lysozyme molecules at the very early stage of the crystallization process by small-angle X-ray scattering

Марченкова, М. А.; Волков, В. В.; Благов, А. Е.; Dyakova, Yu. A.; Ilina, Kseniia B.; Tereschenko, E. Yu.; Тимофеев, В. И.; Pisarevsky, Yu. V.; Kovalchuk, M. V.

doi:10.1134/s1063774516010144

Cited by 29 publications

(16 citation statements)

References 16 publications

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“…The third reason is a nonspecific protein interaction that may not result in aggregation. It can be dependent on the buffer composition (Borshchevskiy et al, 2010;Petsev et al, 2000) and/or, in the case of membrane proteins, on the lipid environment (Nikolaev et al, 2017;Borshchevskiy et al, 2010;Ishchenko et al, 2017) and correspond to crystal contacts (Marchenkova et al, 2016;Kovalchuk et al, 2016) and/or intermediate variants of protein interaction during protein crystallization (Petsev et al, 2000;Kazantsev et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Ryzhykau¹,

Orekhov²,

Rulev³

et al. 2021

Acta Cryst Sect D Struct Biol

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Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is critical for the development of drugs. However, obtaining high-resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low-resolution methods small-angle X-ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small-angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane-protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII–NpHtrII) was performed. The influence of the polydispersity of NpSRII–NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.

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Section: Discussionmentioning

confidence: 99%

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Ryzhykau¹,

Orekhov²,

Rulev³

et al. 2021

Acta Cryst Sect D Struct Biol

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“…We investigated three combinations of such ions: when both of them are bound with the monomers, when only Na ions are embedded in the protein structure, and without both Na and Cl ions incorporated into the lysozyme structure (columns 2, 3, and 4 of Table 1, respectively). These precipitant conditions were chosen because, as in our previous work [9], it was found that MD results on lysozyme oligomers stability investigations are consistent with the SAXS experiments [5,6] only when accounting for all precipitant ions associated with the protein.…”

Section: Resultsmentioning

confidence: 96%

“…Some researchers assumed that it consists only of monomers [4], but most agreed that oligomers are formed in solution. In recent works [5][6][7], it was found that, before its crystallization in solution, lysozyme's oligomers are formed, which are involved in the subsequent growth of the crystal. The authors of this study proposed to use the molecular dynamics (MD) method to assess the stability of oligomers and to study the behavior of atoms and bonds in their structure [8,9].…”

Section: Introductionmentioning

confidence: 99%

Free Energy Change during the Formation of Crystalline Contact between Lysozyme Monomers under Different Physical and Chemical Conditions

et al. 2021

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We use the MM/GBSA method to calculate the free energies of dimer formation by binding two monomers with different combinations of precipitant ions, both embedded in the structure of monomers and in the crystallization solution. It shows that the largest difference in free energy values corresponds to the most accurate dimer model, which considers all precipitant ions in their structure. In addition, it shows that in the absence of precipitant ions in the solution of lysozyme molecules, a monomer is a more energetically favorable state.

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“…In a series of papers [5][6][7], it has recently been established that the addition of precipitants to protein solutions proteins leads to the formation of precursor clusters representing 3D fragments of the crystal structure. In the case of tetragonal modification of lysozyme, crystal octamers are such clusters that are the units of growth of single crystals.…”

Section: Introductionmentioning

confidence: 99%

The Role of Cations of the Precipitant in the Interaction of Protein Molecules in the Lysozyme Oligomers in Crystallization Solutions

et al. 2021

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At the moment, the main opinion is that protein crystallization depends mainly on the the precipitant anions, therefore, there have been only few works devoted to the problem of the influence of its cations. Using the molecular dynamics method, we investigated the stability, changes in the compactness and structural transformations of lysozyme dimers and octamers in solutions with different precipitants (LiCl, NaCl, KCl and CuCl2) in order to study the contribution of cations during crystal formation in more detail. As a result, we found that cations have a rather noticeable effect on the behavior of oligomers: the higher the atomic mass of the cation, the greater the changes in the dimers structures during its dynamics and, according to the data of SAXS experiments, the lower the concentration of dimers. However, for octamers, this dependence is more complicated.

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In situ study of the state of lysozyme molecules at the very early stage of the crystallization process by small-angle X-ray scattering

Cited by 29 publications

References 16 publications

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Ambiguities in and completeness of SAS data analysis of membrane proteins: the case of the sensory rhodopsin II–transducer complex

Free Energy Change during the Formation of Crystalline Contact between Lysozyme Monomers under Different Physical and Chemical Conditions

The Role of Cations of the Precipitant in the Interaction of Protein Molecules in the Lysozyme Oligomers in Crystallization Solutions

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