Ладохин А.С. Молекулярно-биологические аспекты белок-мембранных взаимодействий: термодинамический и кинетический контроль встраивания. – Рукопись.
Диссертация на соискание научной степени доктора биологических наук по специальности 03.00.03 – молекулярная биология. - Институт молекулярной биологии и генетики НАН Украины, Киев, 2001.
Диссертация посвящена установлению общих принципов и молекулярных механизмов спонтанного встраивания белков в мембраны и приобретения ими активной конформации. Предложена и обоснована структурно-термодинамическая концепция белкового встраивания, согласно которой оно обязательно проходит через фолдинговый метастабильный интермедиат. Изучены взаимоотношения кинетического и термодинамического контроля, осуществляемых на разных стадиях встраивания и фолдинга мембранных белков. Доказано, что белки взаимодействуют с поверхностной зоной мембраны в отсутствие аддитивности электростатических и гидрофобных взаимодействий. Количественно охарактеризована энергетическая сопряженность процесса взаимодействия белок–поверхностная зона мембраны с формированием вторичной структуры белка (α-спираль и β-складка). Установлено, что разные типы вторичной структуры имеют близкие значения свободных энергий фолдинга. Разработан, усовершенствован и внедрен в научно-исследовательскую практику ряд флюоресцентных методов изучения термодинамических, кинетических и структурно-функциональных характеристик белково-мембранных взаимодействий.
Ключевые слова: встраивание белков в мембраны, термодинамический и кинетический контроль, фолдинг, электростатические и гидрофобные взаимодействия,
сопряжение связывания и сворачивания, флюоресценция.
Ladokhin A.S. Molecular biology aspects of protein-membrane interactions: thermodynamic and kinetic control of insertion – Manuscript.
Thesis for the Doctor of sciences’ degree by specialty 03.00.03 – molecular biology. – Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, Kyiv, 2001.
Membrane proteins are abundant and important for a variety of cellular functions, yet very little is understood of the molecular events accompanying their assembly [Engelman (1996) Science 274:1850; Bibi (1998) Trends Biochem. Sci. 23:51]. The main goal of this study was to gain insight into thermodynamic and kinetic control of membrane protein folding and stability by studying spontaneously inserting proteins and peptides.In the initial stage of the study we developed new theoretical and experimental approaches, which allowed us to use fluorescence spectroscopy to answer fundamental structural and thermodynamic questions of protein-membrane interactions. We have developed and implemented procedures and experimental schemes to minimize and correct for artifacts arising from the high light scattering caused by lipid vesicles. We have developed a distribution analysis technique to analyze depth-dependent fluorescence quenching in membranes and demonstrated the dynamic nature of this process. We have also developed tools to analyze the mechanism of membrane permeabilization by host-defense peptides. Next we characterize and quantitate the energetics of various steps of membrane binding and insertion of proteins. The latter partition into lipid bilayers mainly through hydrophobic and electrostatic interactions. We have explored systematically free energy group additivity for hydrophobic partitioning and additivity of hydrophobic and electrostatic free energy components. We employed several series of mutants of the proline-rich antimicrobial peptide indolicidin that cannot adopt an ordered secondary structure. The free energy of partitioning into zwitterionic phosphocholine vesicles (ΔGHF) measured by equilibrium dialysis coincides within the experimental error with the prediction of the pentapeptide-based interfacial hydrophobicity scale [Wimley & White (1996) Nature Str. Biol. 3:842]. This confirms the complete group additivity for ΔGHF derived from the pentapeptide scale in the context of a different peptide family. In contrast, we find no additivity in the contributions from hydrophobic and electrostatic interactions: ΔG of transfer of indolicidin mutants into mixed POPC/POPG bilayers depends linearly on bilayer surface potential, but the effective charge of the peptide, Zeff, correlates reciprocally with the peptide’s hydrophobicity, ΔGHF. An empirical rule based on the indolicidin results suggests a reduction of approximately 20% of the value of Zeff for every 3 kcal/mole in ΔGHF. We have determined the energetics of melittin helix formation through measurements of the partitioning free energies and the helicities of native melittin and of a diastereomeric analog with four D-amino acids (D4,L-melittin). Because D4,L-melittin has little secondary structure in either the free or bound forms, it serves as a model for the experimentally inaccessible unfolded bound form of native melittin. The partitioning of native melittin into large unilamellar phosphocholine vesicles is 5.0 ± 0.7 kcal/mol more favorable than that of D4,L-melittin. Differences in the circular dichroism spectra of the two forms of melittin indicate that bound native melittin is more helical than bound D4,L-melittin by about 12 residues. These findings disclose that the free energy reduction per residue accompanying the folding of melittin in membrane interfaces is about 0.4 kcal/mole, consistent with the hypothesis that hydrogen bonding reduces the high cost of partitioning peptide bonds. A similar value of 0.5 kcal/mol per residue has been observed for β-sheet formation by a hexapeptide model system. These two values allow estimation of the energetic consequences of membrane-induced secondary structure formation. We have characterized kinetic control of membrane penetration of cytochrome b5 and melittin. In both cases we find that final insertion is preceded by formation of a metastable intermediate and that heating the system helps to overcome the kinetic barrier towards the final state. We have characterized the conformational change associated with this transition by means of distribution analysis of fluorescence spectroscopy and oriented circular dichroism. For cytochrome b5 , heating was shown to decrease dramatically the tightness of packing of the nonpolar domain, which allows a translocation of the C-terminus across the bilayer. This change is connected to temperature-induced conversion of the exchangeable (“loosely” bound) form of cytochrome b5 to the non-exchangeable (“tightly” bound) form, forms which have dramatically different rates of exchange between membranes We have demonstrated that for melittin an intermediate state has an interfacial topology, while a final pore-forming state consists of transmembrane helices. Kinetic differences for melittin binding/insertion into membranes formed with anionic and zwitterionic lipids result in variation in the mechanism of membrane permeabilization, and thus are important for the peptide’s functioning. We summarize our results in a general model for post-translational membrane insertion of proteins that requires a passage through an obligatory unfolded intermediate state. We suggest that transition from the water-soluble state to this interfacial intermediate is under thermodynamic control, while the final stages of insertion are sometimes irreversible and influenced by kinetic control.
Key words: membrane insertion of proteins, thermodynamic and kinetic control, folding, electrostatic and hydrophobic interactions, fluorescence.
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