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1.
Flavodoxins are well known one-domain alpha/beta electron-transfer proteins that, according to the presence or absence of a approximately 20-residue loop splitting the fifth beta-strand of the central beta-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split beta-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see Lopez-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernandez-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184-47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones.  相似文献   

2.
The secondary structures of the synthetic DNA fragments d(CGCGCGTTTTTCGCGCG) (T5), d(CGCGCGAAAAACGCGCG) (A5), d(CGCGCGTACGCGCG) (TA), and d(CGCGCGATCGCGCG) (AT) were investigated in a combined electrophoretic and spectroscopic study. All the oligomers exist, at low temperature and over a wide range of ionic strength (0.5-100 mM salt) and of nucleotide concentration [0.1-2.0 mM (phosphate)], as a mixture of two slowly interconverting species, identified as the dimeric duplex and the monomeric hairpin structure. The thermodynamic parameters for hairpin denaturation of T5, A5, TA, and AT and for duplex denaturation of d(CGCGCG) show that (a) the hairpins are more stable than the reference hexamer duplex at all accessible nucleotide concentrations; (b) the loop contributes favorably to the enthalpy change of hairpin denaturation in the four DNA fragments; (c) the base composition of the loop (A vs T) and the size of the loop (A5/T5 vs TA/AT) do not appreciably influence the enthalpic contents of the hairpins; (d) hairpins TA and AT, with two AT bases intervening in the CG self-complementary part of the molecule, exhibit a markedly higher thermal stability than hairpins T5 and A5, which is entropic in origin. These findings are consistent with the presence of two-residue loops in the tetradecamers TA and AT.  相似文献   

3.
To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37+/-3 A at 3 M guanidine hydrochloride. F?rster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol(-1). The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9 degrees C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.  相似文献   

4.
Bollen YJ  Sánchez IE  van Mierlo CP 《Biochemistry》2004,43(32):10475-10489
The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.  相似文献   

5.
6.
Protein intermediates in equilibrium with native states may play important roles in protein dynamics but, in cases, can initiate harmful aggregation events. Investigating equilibrium protein intermediates is thus important for understanding protein behaviour (useful or pernicious) but it is hampered by difficulties in gathering structural information. We show here that the phi-analysis techniques developed to investigate transition states of protein folding can be extended to determine low-resolution three-dimensional structures of protein equilibrium intermediates. The analysis proposed is based solely on equilibrium data and is illustrated by determination of the structure of the apoflavodoxin thermal unfolding intermediate. In this conformation, a large part of the protein remains close to natively folded, but a 40 residue region is clearly unfolded. This structure is fully consistent with the NMR data gathered on an apoflavodoxin mutant designed specifically to stabilise the intermediate. The structure shows that the folded region of the intermediate is much larger than the proton slow-exchange core at 25 degrees C. It also reveals that the unfolded region is made of elements whose packing surface is more polar than average. In addition, it constitutes a useful guide to rationally stabilise the native state relative to the intermediate state, a far from trivial task.  相似文献   

7.
Titin (first known as connectin) is a vast modular protein found in vertebrate striated muscle. It is thought to assist myofibrillogenesis and to provide a passive elastic restoring force that helps to keep the thick filaments properly centered in the sarcomere. We show that representative titin modules do indeed fold independently, and report their stabilities (i.e., delta G of unfolding and melting temperature) as measured by circular dichroism, fluorescence, and nuclear magnetic resonance spectroscopies. We find that there is a region-dependent variation in stability, although we find no evidence to support a proposed elastic mechanism based on a molten-globular-like equilibrium folding intermediate, nor do our calculations support any mechanism based on the configurational entropy of the molecule itself; instead we suggest a model based on hydrophobic hinge regions that would not be strongly dependent on the precise folding pattern of the chain.  相似文献   

8.
Group II introns: structure, folding and splicing mechanism   总被引:4,自引:0,他引:4  
Group II introns are large autocatalytic RNAs found in organellar genomes of plants and lower eukaryotes, as well as in some bacterial genomes. Interestingly, these ribozymes share characteristic traits with both spliceosomal introns and non-LTR retrotransposons and may have a common evolutionary ancestor. Furthermore, group II intron features such as structure, folding and catalytic mechanism differ considerably from those of other large ribozymes, making group II introns an attractive model system to gain novel insights into RNA biology and biochemistry. This review explores recent advances in the structural and mechanistic characterization of group II intron architecture and self-splicing.  相似文献   

9.
10.
The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD) simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an "activated" form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins.  相似文献   

11.
Conformational stability and mechanism of folding of ribonuclease T1   总被引:5,自引:0,他引:5  
Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.  相似文献   

12.
It is believed that the critical step in the pathogenesis of transmissible spongiform encephalopathies is a transition of prion protein (PrP) from an alpha-helical conformation, PrP(C), to a beta-sheet-rich form, PrP(Sc). Native prion protein contains a single disulfide bond linking Cys residues at positions 179 and 214. To elucidate the role of this bridge in the stability and folding of the protein, we studied the reduced form of the recombinant human PrP as well as the variant of PrP in which cysteines were replaced with alanine residues. At neutral pH, the reduced prion protein and the Cys-free mutant were insoluble and formed amorphous aggregates. However, the proteins could be refolded in a monomeric form under the conditions of mildly acidic pH. Spectroscopic experiments indicate that the monomeric Cys-free and reduced PrP have molten globule-like properties, i.e. they are characterized by compromised tertiary interactions, an increased exposure of hydrophobic surfaces, lack of cooperative unfolding transition in urea, and partial loss of native (alpha-helical) secondary structure. In the presence of sodium chloride, these partially unfolded proteins undergo a transition to a beta-sheet-rich structure. However, this transition is invariably associated with protein oligomerization. The present data argue against the notion that reduced prion protein can exist in a stable monomeric form that is rich in beta-sheet structure.  相似文献   

13.
Alanine substitutions were made for 15 amino acids in the cytoplasmic loop between transmembrane helices 6 and 7 (L6/7) of the human alpha(1)-subunit of Na,K-ATPase. Most mutations reduced Na,K-ATPase activity by less than 50%; however, the mutations R834A, R837A, and R848A reduced Na,K-ATPase activity by 75, 89, and 66%, respectively. Steady-state phosphoenzyme formation from ATP was reduced in mutants R834A, R837A, and R848A, and R837A also had a faster E(2)P --> E(2) dephosphorylation rate compared with the wild-type enzyme. Effects of L6/7 mutations on the phosphorylation domain of the protein were also demonstrated by (18)O exchange, which showed that intrinsic rate constants for P(i) binding and/or reaction with the protein were altered. Although most L6/7 mutations had no effect on the interaction of Na(+) or K(+) with Na,K-ATPase, the E825A, E828A, R834A, and R837A mutations reduced the apparent affinity of the enzyme for both Na(+) and K(+) by 1.5-3-fold. 1-Bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU(3+)), a competitive antagonist of Rb(+) and Na(+) occlusion, was used to test whether charged residues in L6/7 are involved in binding monovalent cations and cation antagonists. Br-TITU(3+) inhibited ouabain binding to wild type Na,K-ATPase with an IC(50) of 30 microM. Ouabain binding to the E825A, E828A, R834A, or R837A mutants was still inhibited by Br-TITU(3+), indicating that Br-TITU(3+) does not bind to charged residues in L6/7. This observation makes it unlikely that L6/7 functions as a cytoplasmic cation binding site in Na,K-ATPase, and together with the effects of L6/7 mutations on phosphate interactions with the enzyme suggests that L6/7 is important in stabilizing the phosphorylation domain and its relationship to the ion binding sites of the protein.  相似文献   

14.
15.
16.
17.
The ratio of short to long loop nephrons (SLNs and LLNs, respectively) in laboratory rodents (mice, rats, hamsters, gerbils, and guinea pigs) was investigated using the air cast method. In mice and rats, the percentage of SLNs was significantly higher than that of LLNs, while in hamsters and gerbils, the reverse was true (% of LLNs >% of SLNs). In guinea pigs, no significant difference in the percentages of LLNs and SLNs was noted.  相似文献   

18.
19.
The simplicity of C. elegans makes it an outstanding system to study the role of Wnt signaling in development. Many asymmetric cell divisions in C. elegans require the Wnt/beta-catenin asymmetry pathway. Recent studies confirm that SYS-1 is a structurally and functionally divergent beta-catenin, and implicate lipids and retrograde trafficking in maintenance of WRM-1/beta-catenin asymmetry. Wnts also regulate short-range events such as spindle rotation and gastrulation, and a PCP-like pathway regulates asymmetric divisions. Long-range, cell non-autonomous Wnt signals regulate vulval induction. Both short-range and long-range Wnt signal s are regulated by recycling of MIG-14/Wntless via the retromer complex. These studies indicate that C. elegans continues to be useful for identifying new, conserved mechanisms underlying Wnt signaling in metazoans.  相似文献   

20.
Mick V  Geister S  Paulsen H 《Biochemistry》2004,43(46):14704-14711
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.  相似文献   

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