Search results for "protein folding"
showing 10 items of 196 documents
Site-Specific Information on Membrane Protein Folding by Electron Spin Echo Envelope Modulation Spectroscopy
2010
Compared to folding of soluble proteins, folding of membrane proteins is complicated by the fact that it requires an amphiphilic environment. Few existing techniques can provide structurally resolved information on folding kinetics. For the major plant light harvesting complex LHCII, it is demonstrated that changes in water accessibility of a particular amino acid residue can be followed during folding by measuring the hyperfine interaction of spin labels with deuterium nuclei of heavy water. The incorporation of residue 196 into the hydrophobic core of a detergent micelle was investigated. The technique provides a time constant that is similar to the one found with fluorescence spectroscop…
Folding in vitro of light-harvesting chlorophyll a/b protein is coupled with pigment binding.
2002
The major light-harvesting chlorophyll a/b protein (LHCIIb) of the plant photosynthetic apparatus is able to self-organise in vitro. When the recombinant apoprotein, Lhcb1, is solubilised in the denaturing detergent sodium (or lithium) dodecylsulfate (SDS or LDS) and then mixed with chlorophylls and carotenoids under renaturing conditions, structurally authentic LHCIIb forms. Assembly of functional LHCIIb, as indicated by the establishment of energy transfer between complex-bound chlorophyll molecules, occurs in two apparent kinetic steps with time constants of 10 to 30 seconds and 50 to 300 seconds, depending on the reaction conditions. Here, we use circular dichroism (CD) in the far-UV ra…
Random mutations directed to transmembrane and loop domains of the light-harvesting chlorophyll a/b protein: impact on pigment binding.
1999
The major light-harvesting complex of photosystem II (LHCII) can be reconstituted in vitro by folding its bacterially expressed apoprotein, Lhcb, in detergent solution in the presence of chlorophylls and carotenoids. To compare the impact of alpha-helical transmembrane domains and hydrophilic loop domains of the apoprotein on complex formation and stability, we introduced random mutations into a segment of the protein comprising the stromal loop, the third (C-proximal) transmembrane helix, and part of the amphipathic helix in the C-terminal domain. The mutant versions of Lhcb were screened for the loss of their ability to form stable LHCII upon reconstitution in vitro. Most steps during the…
Decreasing the chlorophyll a/b ratio in reconstituted LHCII: Structural and functional consequences
1999
Trimeric (bT) and monomeric (bM) light-harvesting complex II (LHCII) with a chlorophyll a/b ratio of 0.03 were reconstituted from the apoprotein overexpressed in Escherichia coli. Chlorophyll/xanthophyll and chlorophyll/protein ratios of bT complexes and 'native' LHCII are rather similar, namely, 0.28 vs 0. 27 and 10.5 +/- 1.5 vs 12, respectively, indicating the replacement of most chlorophyll a molecules with chlorophyll b, leaving one chlorophyll a per trimeric complex. The LD spectrum of the bT complexes strongly suggests that the chlorophyll b molecules adopt orientations similar to those of the chlorophylls a that they replace. The circular dichroism (CD) spectra of bM and bT complexes…
Filling the “green gap” of the major light-harvesting chlorophyll a/b complex by covalent attachment of Rhodamine Red
2009
AbstractThe major light-harvesting chlorophyll a/b complex (LHCII) greatly enhances the efficiency of photosynthesis in green plants. Recombinant LHCII can be assembled in vitro from its denatured, bacterially expressed apoprotein and plant pigments. This makes it an interesting candidate for biomimetic light-harvesting in photovoltaic applications. Due to its almost 20 pigments bound per apoprotein, LHCII absorbs efficiently in the blue and red spectral domains of visible light but less efficiently in the green domain, the so-called “green gap” in its absorption spectrum. Here we present a hybrid complex of recombinant LHCII with organic dyes that add to LHCII absorption in the green spect…
Early folding events during light harvesting complex II assembly in vitro monitored by pulsed electron paramagnetic resonance
2016
Efficient energy transfer in the major light harvesting complex II (LHCII) of green plants is facilitated by the precise alignment of pigments due to the protein matrix they are bound to. Much is known about the import of the LHCII apoprotein into the chloroplast via the TOC/TIC system and its targeting to the thylakoid membrane but information is sparse about when and where the pigments are bound and how this is coordinated with protein folding. In vitro, the LHCII apoprotein spontaneously folds and binds its pigments if the detergent-solubilized protein is combined with a mixture of chlorophylls a and b and carotenoids. In the present work, we employed this approach to study apoprotein fo…
The Folding State of the Lumenal Loop Determines the Thermal Stability of Light-Harvesting Chlorophyll a/b Protein
2004
The major light-harvesting protein of photosystem II (LHCIIb) is the most abundant chlorophyll-binding protein in the thylakoid membrane. It contains three membrane-spanning alpha helices; the first and third one closely interact with each other to form a super helix, and all three helices bind most of the pigment cofactors. The protein loop domains connecting the alpha helices also play an important role in stabilizing the LHCIIb structure. Single amino acid exchanges in either loop were found to be sufficient to significantly destabilize the complex assembled in vitro [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093. Mick, V., Eggert, K., Heinemann, B., Geister, S., and…
The Light-Harvesting Chlorophyll a/b Complex Can Be Reconstituted in Vitro from Its Completely Unfolded Apoprotein
2003
The major light-harvesting chlorophyll a/b protein (LHCIIb) of higher plants is one of the few membrane proteins that can be refolded in vitro. During folding, the apoprotein is assembled with pigments to form a structurally authentic and functional pigment--protein complex. All reconstitution procedures used so far include solubilization of the apoprotein in sodium dodecyl sulfate (SDS) where the protein adopts approximately half of its alpha-helical folding present in the native structure. This paper shows that this preformed alpha-helix is not a prerequisite for LHCIIb folding in vitro. The apoprotein can also be reconstituted starting from a solution in guanidinium hydrochloride (Gnd) w…
Glucagon fibril polymorphism reflects differences in protofilament backbone structure
2010
Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril class…
Calorimetric and structural investigation of the interaction between bovine serum albumin and high molecular weight dextran in water.
2005
This work studies specific interactions between a small globular protein and a highly flexible, branched polysaccharide using differential scanning calorimetry (DSC), circular dichroism (CD), fluorescence, and turbidimetry measurements. It uses the system water/bovine serum albumin (BSA)/dextran (D 2000) as a model. Dextran molecules are able to form interpolymeric complexes with BSA in water at both low and high temperatures if the polysaccharide is in excess and if the protein exists in its associated state. It leads to a partial destabilization of the secondary and tertiary structures of the protein and an additional exposure of the hydrophobic tryptophan residues to the surface of globu…