Folding in vitro of light-harvesting chlorophyll a/b protein is coupled with pigment binding

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 range to monitor the folding of the LHCIIb apoprotein as it is complexed with pigments. The alpha-helix content in the protein's secondary structure increases in two apparent kinetic steps with time constants similar to those observed for the establishment of chlorophyll energy transfer. When the carotenoid concentration in the reaction mixture is reduced, the time constants of alpha-helix formation increase, as do those for the appearance of chlorophyll energy transfer. This indicates that both processes, pigment assembly and secondary structure formation, are tightly coupled. A substantial amount of alpha-helix is present in dodecylsulfate-solubilised LHCIIb apoprotein and appears to be distributed among various protein domains.     

Consecutive binding of chlorophylls a and b during the assembly in vitro of light-harvesting chlorophyll-a/b protein (LHCIIb)

The apoprotein of the major light-harvesting chlorophyll a/b complex (LHCIIb) is post-translationally imported into the chloroplast, where membrane insertion, protein folding, and pigment binding take place. The sequence and molecular mechanism of the latter steps is largely unknown. The complex spontaneously self-organises in vitro to form structurally authentic LHCIIb upon reconstituting the unfolded recombinant protein with the pigments chlorophyll a, b, and carotenoids in detergent micelles. Former measurements of LHCIIb assembly had revealed two apparent kinetic phases, a faster one (tau1) in the range of 10 s to 1 min, and a slower one (tau2) in the range of several min. To unravel the sequence of events we analysed the binding of chlorophylls into the complex by using time-resolved fluorescence measurements of resonance energy transfer from chlorophylls to an acceptor dye attached to the apoprotein. Chlorophyll a, offered in the absence of chlorophyll b, bound with the faster kinetics (tau1) exclusively whereas chlorophyll b, in the absence of chlorophyll a, bound predominantly with the slower kinetics (tau2). In double-jump experiments, LHCIIb assembly could be dissected into a faster chlorophyll a and a subsequent, predominantly slower chlorophyll b-binding step. The assignment of the faster and the slower kinetic phase to predominantly chlorophyll a and exclusively chlorophyll b binding, respectively, was verified by analysing the assembly kinetics with a circular dichroism signal in the visible domain presumably reflecting the establishment of pigment-pigment interactions. We propose that slow chlorophyll binding is confined to the exclusively chlorophyll b binding sites whereas faster binding occurs to the chlorophyll a binding sites. The latter sites can bind both chlorophylls a and b but in a reversible fashion as long as the complex is not stabilised by proper occupation of the chlorophyll b sites. The resulting two-step model of LHCIIb assembly is able to reconcile the highly specific binding sites containing either chlorophyll a or b, as seen in the recent crystal structures of LHCIIb, with the observation of promiscuous binding sites able to bind both chlorophyll a and b in numerous reconstitution analyses of LHCIIb assembly.     

Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the mutant protein with pigments in vitro. The resulting collection of mutated recombinant LHCIIb versions was screened by using a 96-well-format plate-based procedure described previously [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093], enabling us to test several thousand mutants for their ability to form stable pigment-protein complexes in vitro. At least one-third of the positions in the loop domain turned out to be sensitive targets; i.e., their exchange abolished formation of LHCIIb in vitro. This confirms our earlier notion that the LHCIIb loop domains contribute more specifically to complex formation and/or stabilization than by merely connecting the alpha helices. Among the target sites, glycines and hydrophilic amino acids are more prominently represented than hydrophobic ones. Specifically, the exchange of any of the three acidic amino acids in the lumenal loop abolishes reconstitution of stable pigment-protein complexes, suggesting that ionic interactions with other protein domains are important for correct protein folding or complex stabilization. One hydrophobic amino acid, tryptophan in position 97, has been hit repeatedly in independent mutation experiments. From the LHCIIb structure and previous mutational analyses, we propose a stabilizing interaction between this amino acid and F195 near the C-proximal end of the third transmembrane helix.     

Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex

The major light-harvesting complex (LHCIIb) of photosystem II can be reconstituted in vitro from its recombinant apoprotein in the presence of a mixture of carotenoids and chlorophylls a and b. By varying the chlorophyll a/b ratio in the reconstitution mixture, the relative amounts of chlorophyll a and chlorophyll b bound to LHCIIb can be changed. We have analyzed the chlorophyll stoichiometry in recombinant wild type and mutant LHCIIb reconstituted at different chlorophyll a/b ratios in order to assess relative affinities of the chlorophyll-binding sites. This approach reveals five sites that exclusively bind chlorophyll b. Another site exhibits a slight preference of chlorophyll b over chlorophyll a. The remaining six sites are filled preferentially with chlorophyll a but also tolerate chlorophyll b when this is offered at a large excess. Three of these chlorophyll a-affine sites could be assigned to distinct positions defined by the three-dimensional LHCIIb structure. Exclusive chlorophyll b sites complemented by chlorophyll a sites that are selective only to a certain extent are consistent with the observation that chlorophyll b but not chlorophyll a is essential for reconstituting stable LHCIIb. These data offer an explanation why a rather constant chlorophyll a/b ratio is observed in native LHCIIb despite the apparent promiscuity of some binding sites.     

Early steps in the assembly of light-harvesting chlorophyll a/b complex: time-resolved fluorescence measurements

The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.     

Effects of chlorophyll a, chlorophyll b, and xanthophylls on the in vitro assembly kinetics of the major light-harvesting chlorophyll a/b complex, LHCIIb

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem II in higher plants can be reconstituted with pigments in lipid-detergent micelles. The pigment-protein complexes formed are functional in that they perform efficient internal energy transfer from chlorophyll b to chlorophyll a. LHCIIb formation in vitro, can be monitored by the appearance of energy transfer from chlorophyll b to chlorophyll a in time-resolved fluorescence measurements. LHCIIb is found to form in two apparent kinetic steps with time constants of about 30 and 200 seconds. Here we report on the dependence of the LHCIIb formation kinetics on the composition of the pigment mixture used in the reconstitution. Both kinetic steps slow down when the concentration of either chlorophylls or carotenoids is reduced. This suggests that the slower 200 seconds formation of functional LHCIIb still includes binding of both chlorophylls and carotenoids. LHCIIb formation is accelerated when the chlorophylls in the reconstitution mixture consist predominantly of chlorophyll a although the complexes formed are thermally less stable than those reconstituted with a chlorophyll a:b ratio < or = 1. This indicates that although chlorophyll a binding is more dominant in the observed rate of LHCIIb formation, the occupation of (some) chlorophyll binding sites with chlorophyll b is essential for complex stability. The accelerating effect of various carotenoids (lutein, zeaxanthin, violaxanthin, neoxanthin) on LHCIIb formation correlates with their affinity to two lutein-specific binding sites. We conclude that the occupation of these two carotenoid binding sites but not of the third (neoxanthin-specific) binding site is an essential step in the assembly of LHCIIb in vitro.     

The folding state of the lumenal loop determines the thermal stability of light-harvesting chlorophyll a/b protein

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 Paulsen, H (2004) Biochemistry 43, 5467-5473]. This work presents an analysis of such point mutations in the lumenal loop with regard to the extent and nature of their effect on LHCIIb stability to obtain detailed information on the contribution of this loop to stabilizing the complex. Most of the mutant proteins yielded pigment-protein complexes if their reconstitution and/or isolation was performed under mild conditions; however, the yields were significantly different. Several mutations in the vicinity of W97 in the N-proximal section of the loop gave low reconstitution yields even under very mild conditions. This confirms our earlier notion that W97 may be of particular relevance in stabilizing LHCIIb. The same amino acid exchanges accelerated thermal complex dissociation in the absence of lithium dodecyl sulfate (LDS) and raised the accessibility of the lumenal loop to protease; both effects were well correlated with the reduction in reconstitution yields. We conclude that a detachment of the lumenal loop is a possible first step in the dissociation of LHCIIb. Dramatically reduced complex yields in the presence but not in the absence of LDS were observed for some but not all mutants, particularly those near the C-proximal end of the loop. We conclude that complex stabilities in the absence and in the presence of LDS do not correlate and most likely are determined by different structural characteristics, at least in LHCIIb but maybe also in other membrane proteins.     

The binding of Xanthophylls to the bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-end group

The pigment composition of the light-harvesting complexes (LHCs) of higher plants is highly conserved. The bulk complex (LHCIIb) binds three xanthophyll molecules in combination with chlorophyll (Chl) a and b. The structural requirements for binding xanthophylls to LHCIIb have been examined using an in vitro reconstitution procedure. Reassembly of the monomeric recombinant LHCIIb was performed using a wide range of native and nonnative xanthophylls, and a specific requirement for the presence of a hydroxy group at C-3 on a single beta-end group was identified. The presence of additional substituents (e.g. at C-4) did not interfere with xanthophyll binding, but they could not, on their own, support reassembly. cis isomers of zeaxanthin, violaxanthin, and lutein were not bound, whereas all-trans-neoxanthin and different chiral forms of lutein and zeaxanthin were incorporated into the complex. The C-3 and C-3' diols lactucaxanthin (a carotenoid native to many plant LHCs) and eschscholtzxanthin (a retro-carotenoid) both behaved very differently from lutein and zeaxanthin in that they would not support complex reassembly when used alone. Lactucaxanthin could, however, be bound when lutein was also present, and it showed a high affinity for xanthophyll binding site N1. In the presence of lutein, lactucaxanthin was readily bound to at least one lutein-binding site, suggesting that the ability to bind to the complex and initiate protein folding may be dependent on different structural features of the carotenoid molecule. The importance of carotenoid end group structure and ring-to-chain conformation around the C-6-C-7 torsion angle of the carotenoid molecule in binding and complex reassembly is discussed.     

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.     

Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.     

Unfolding and refolding of hen egg-white riboflavin binding protein

The unfolding and refolding of riboflavin-binding protein (RfBP) from hen egg-white induced by addition of guanidinium chloride (GdnHCl), and its subsequent removal by dialysis have been studied by c.d. and fluorescence for both the native and reduced protein. The reduction of its nine disulphide bonds causes a reduction in the secondary structure (alpha-helix plus beta-sheet) from 63% to 33% of the amino acid residues. Unfolding of the native protein occurred in two phases; the first involving a substantial loss of tertiary structure, followed by a second phase involving loss of secondary structure at higher GdnHCl concentrations. By contrast this biphasic behaviour was not discernible in the reduced protein. The loss of ability to bind riboflavin occurred after the first phase of unfolding. Comparison of unfolding of the holoprotein and apoprotein suggested that riboflavin has only a small stabilizing effect on the unfolding process. After removal of GdnHCl, the holoprotein, apoprotein and reduced protein assumed their original conformation. The significance of the results in relation to various models for protein folding is discussed.     

Stability of the glycerol facilitator in detergent solutions

Understanding membrane protein folding and stability is required for a molecular explanation of function and for the development of interventions in membrane protein folding diseases. Stable aqueous detergent solutions of the Escherichia coli glycerol facilitator in its native oligomeric state have been difficult to prepare as the protein readily unfolds and forms nonspecific aggregates. Here, we report a study of the structure and stability of the glycerol facilitator in several detergent solutions by Blue Native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD), and fluorescence. Protein tetramers were prepared in neutral dodecyl maltoside (DDM) and in zwitterionic lysomyristoylphosphatidylcholine (LMPC) detergent solutions that are stable during SDS-PAGE. Thermal unfolding experiments show that the protein is more stable in LMPC than in DDM. Tertiary structure unfolds before quaternary and some secondary structure in LMPC, whereas unfolding is more cooperative in DDM. The high stability of the protein in DDM is evident from the unfolding half-life of 8 days in 8 M urea, suggesting that hydrophobic interactions contribute to the stability. The protein unfolds readily in LMPC below pH 6, whereas the tetramer remains intact at pH 4 in DDM. At pH 4 in DDM, the protein is more sensitive than at neutral pH to unfolding by SDS and the effect is reversible. At pH 3 in DDM, the tetramer unfolds, losing its tertiary structure but retaining native helical structure which melts at significantly lower temperatures than in the native tetramer. The glycerol facilitator prepared in SDS is mainly monomeric and has ~10% less alpha-helix than the native protein. CD suggests that it forms a condensed structure with non-native tertiary contacts highly similar to the state observed in LMPC at low pH. The implications of the results for in vitro and in vivo folding of the protein are discussed.     

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.     

Random mutations directed to transmembrane and loop domains of the light-harvesting chlorophyll a/b protein: impact on pigment binding.

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 screening, including expression of the recombinant protein, its reconstitution with pigments, and the assay for complex formation by measuring energy transfer from chlorophyll b to chlorophyll a, were performed as one-vessel reactions on 96-well microtiter plates. This enabled us to screen several hundred mutant Lhcb versions. Mutants that had lost their ability to form stable LHCII carried between one and four amino acid exchanges. Among the single-point mutations, several were at positions in the C-proximal transmembrane helix, including an amino acid that is thought to be directly involved in chlorophyll binding. However, we also found four point mutations in the stromal loop domain that, in our assay, completely abolished the formation of stable LHCII. These data show that the stromal loop domain has a significant impact on LHCII formation and/or stability in vitro.     

Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling

Paramagnetic relaxation has been used to monitor the formation of structure in the folding peptide chain of guanidinium chloride-denatured acyl-coenzyme A-binding protein. The spin label (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSL) was covalently bound to a single cysteine residue introduced into five different positions in the amino acid sequence. It was shown that the formation of structure in the folding peptide chain at conditions where 95% of the sample is unfolded brings the relaxation probe close to a wide range of residues in the peptide chain, which are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded state, and that these may be of importance to the initiation of protein folding.     

Iron-sulfur cluster biosynthesis. Thermatoga maritima IscU is a structured iron-sulfur cluster assembly protein

Genetic evidence has indicated that Isc proteins play an important role in iron-sulfur cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent iron-sulfur cluster that is subsequently delivered to target iron-sulfur apoproteins. We report the characterization of an IscU from Thermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. M?ssbauer (delta = 0.29 +/- 0.03 mm/s, DeltaE(Q) = 0.58 +/- 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]2+ cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% alpha-helix, 13.1% antiparallel beta-sheet, 11.3% parallel beta-sheet, 20.2% beta-turn, and 19.1% other at 20 degrees C, with negligible spectral change observed at 70 degrees C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in 1H-15N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritima IscU-mediated reconstitution of human apoferredoxin.     

Protein structures in SDS micelle-protein complexes.

Sodium dodecyl sulfate (SDS) is used more often than any other detergent as an excellent denaturing or "unfolding" detergent. However, formation of ordered structure (alpha-helix or beta-sheet) in certain peptides is known to be induced by interaction with SDS micelles. The SDS-induced structures formed by these peptides are amphiphilic, having both a hydrophobic and a hydrophilic face. Previous work in this area has revealed that SDS induces helical folding in a wide variety of non-helical proteins. Here, we describe the interaction of several structurally unrelated proteins with SDS micelles and the correlation of these structures to helical amphiphilic regions present in the primary sequence. It is likely that the ability of native nonordered protein structures to form induced amphiphilic ordered structures is rather common.     

A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins

The NifL PAS domain from Azotobacter vinelandii is a flavoprotein with FAD as the prosthetic group. Here we describe a novel immobilization procedure for the large-scale preparation of apo NifL PAS domain and its efficient reconstitution with either 2,4a-13C-FAD or 2,4a-13C-FMN. In this procedure, the His-tagged holoprotein is bound to an immobilized metal affinity column and the flavin is released by washing the column with buffer containing 2 M KBr and 2 M urea. The apoprotein is reconstituted on-column with the (artificial) flavin cofactor, and then eluted with buffer containing 250 mM imidazole. Alternatively, the immobilized apoprotein can be released from the column matrix before reconstitution.The His-tag based immobilization method of preparing reconstituted (or apo) NifL PAS domain protein has the advantage that it combines a protein affinity chromatography technique with limited protein loss, resulting in a high protein yield with extremely efficient flavin reconstitution. This on-column reconstitution method can also be used in cases where the apoprotein is unstable. Therefore, it may develop as a universal method for replacement of flavin or other cofactors.     

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.     

Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding

Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.