Presence of the cofactor speeds up folding of Desulfovibrio desulfuricans flavodoxin

Flavodoxin is an alpha/beta protein with a noncovalently bound flavin-mononucleotide (FMN) cofactor. The apo-protein adopts a structure identical to that of the holo-form, although there is more dynamics in the FMN-binding loops. The equilibrium unfolding processes of Azotobacter vinelandii apo-flavodoxin, and Desulfovibrio desulfuricans ATCC strain 27774 apo- and holo-flavodoxins involve rather stable intermediates. In contrast, we here show that both holo- and apo-forms of flavodoxin from D. desulfuricans ATCC strain 29577 (75% sequence similarity with the strain 27774 protein) unfold in two-state equilibrium processes. Moreover, the FMN cofactor remains bound to the unfolded holo-protein. The folding and unfolding kinetics for holo-flavodoxin exhibit two-state behavior, albeit an additional slower phase is present at very low denaturant concentrations. The extrapolated folding time in water for holo-flavodoxin, approximately 280 microsec, is in excellent agreement with that predicted from the protein's native-state topology. Unlike the holo-protein behavior, the folding and unfolding reactions for apo-flavodoxin are best described by two kinetic phases, with rates differing approximately 15-fold, suggesting the presence of a kinetic intermediate. Both folding phases for apo-flavodoxin are orders of magnitude slower (40- and 530-fold, respectively) than that for the holo-protein. We conclude that polypeptide-cofactor interactions in the unfolded state of D. desulfuricans strain 29577 flavodoxin alter the kinetic-folding path towards two-state and speed up the folding reaction.     

No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricans flavodoxin

Flavodoxins are proteins with an alpha/beta doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K(D)=0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (DeltaG=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high (K(D)=0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin

Contribution of three regions (phosphate-binding, 50’s and 90’s loops) of Anabaena apoflavodoxin to FMN binding and reduction potential was studied. Thr12 and Glu16 did not influence FMN redox properties, but Thr12 played a role in FMN binding. Replacement of Trp57 with Glu, Lys or Arg moderately shifted E_ox/sq and E_sq/hq and altered the energetic of the FMN redox states binding profile. Our data indicate that the side chain of position 57 does not modulate E_ox/sq by aromatic stacking or solvent exclusion, but rather by influencing the relative strength of the H-bond between the N(5) of the flavin and the Asn58-Ile59 bond. A correlation was observed between the isoalloxazine increase in solvent accessibility and less negative E_sq/hq. Moreover, E_sq/hq became less negative as positively charged residues were added near to the isoalloxazine. Ile59 and Ile92 were simultaneously mutated to Ala or Glu. These mutations impaired FMN binding, while shifting E_sq/hq to less negative values and E_ox/sq to more negative. These effects are discussed on the bases of the X-ray structures of some of the Fld mutants, suggesting that in Anabaena Fld the structural control of both electron transfer steps is much more subtle than in other Flds.     

The reconstitution of unfolded myoglobin with hemin dicyanide is not accelerated by fly-casting

This study explores how the kinetics of a coupled folding/binding reaction depend on the initial conformation of the protein. Stopped-flow spectroscopy is used to monitor the reaction of apo-myoglobin (aMb) with hemin dicyanide at pH 7.2. Different initial aMb conformations are tested. In the case of acid-denatured aMb, the observed kinetics are consistent with a "fly-casting" scenario [Shoemaker et al., Proc. Natl. Acad. Sci. USA 97 (2000) 8868-8873]. However, the formation of a compact complex proceeds more rapidly in the case of prefolded aMb. This finding is opposite to what would be expected based on predictions of the fly-casting model.     

Thermal unfolding of Apo and Holo Desulfovibrio desulfuricans flavodoxin: cofactor stabilizes folded and intermediate states

We here compare thermal unfolding of the apo and holo forms of Desulfovibrio desulfuricans flavodoxin, which noncovalently binds a flavin mononucleotide (FMN) cofactor. In the case of the apo form, fluorescence and far-UV circular dichroism (CD) detected transitions are reversible but do not overlap (T(m) of 50 and 60 degrees C, respectively, pH 7). The thermal transitions for the holo form follow the same pattern but occur at higher temperatures (T(m) of 60 and 67 degrees C for fluorescence and CD transitions, respectively, pH 7). The holoprotein transitions are also reversible and exhibit no protein concentration dependence (above 10 microM), indicating that the FMN remains bound to the polypeptide throughout. Global analysis shows that the thermal reactions for both apo and holo forms proceed via an equilibrium intermediate that has approximately 90% nativelike secondary structure and significant enthalpic stabilization relative to the unfolded states. Incubation of unfolded holoflavodoxin at high temperatures results in FMN dissociation. Rebinding of FMN at these conditions is nominal, and therefore, cooling of holoprotein heated to 95 degrees C follows the refolding pathway of the apo form. However, FMN readily rebinds to the apoprotein at lower temperatures. We conclude that (1) a three-state thermal unfolding behavior appears to be conserved among long- and short-chain, as well as apo and holo forms of, flavodoxins and (2) flavodoxin's thermal stability (in both native and intermediate states) is augmented by the presence of the FMN cofactor.     

蛋白质折叠速率预测研究进展

蛋白质折叠速率预测是当今生物物理学最具挑战性的课题之一。近年来,该领域的研究取得了很大的进展,提出了许多经验参数,例如:接触序、长程序、总接触距离、链拓扑参数、二级结构含量、有效长度、螺旋参数、n-阶接触距离等。这些参数都和蛋白质的折叠速率有很好的相关性,基于这些参数的各种预测方法所得到的预测结果也与实验数据较好地吻合。     

The Escherichia coli alkylation response protein AidB is a redox partner of flavodoxin and binds RNA and acyl carrier protein

Microorganisms are exposed to a wide variety of exogenous and endogenous chemical agents that alkylate DNA. Escherichia coli cells exhibit an adaptive response that recognizes and repairs alkylated DNA lesions using Ada, AlkA, and AlkB enzymes. Another alkylation response protein, the DNA-binding flavoprotein AidB, was proposed to repair DNA or protect it from chemical alkylating agents, but direct evidence for its role is lacking. Here, AidB was shown to form tight complexes with both flavodoxin and acyl carrier protein. In addition, electron transfer between 1-electron and 2-electron reduced flavodoxin to oxidized AidB was observed, although with very small rate constants. AidB was found to bind to RNA, raising the prospect that the protein may have a role in protection of RNA from chemical alkylation. Finally, the reagent N-methyl-N′-nitro-N-nitrosoguanidine was eliminated as a direct substrate of the enzyme.     

Prediction of Protein Structure by Simulating Coarse-grained Folding Pathways: A Preliminary Report

Abstract

A set of software tools designed to study protein structure and kinetics has been developed. The core of these tools is a program called Folding Machine (FM) which is able to generate low resolution folding pathways using modest computational resources. The FM is based on a coarse-grained kinetic ab initio Monte-Carlo sampler that can optionally use information extracted from secondary structure prediction servers or from fragment libraries of local structure. The model underpinning this algorithm contains two novel elements: (a) the conformational space is discretized using the Ramachandran basins defined in the local φ-ψ energy maps; and (b) the solvent is treated implicitly by rescaling the pairwise terms of the non-bonded energy function according to the local solvent environments. The purpose of this hybrid ab initio/knowledge-based approach is threefold: to cover the long time scales of folding, to generate useful 3-dimensional models of protein structures, and to gain insight on the protein folding kinetics. Even though the algorithm is not yet fully developed, it has been used in a recent blind test of protein structure prediction (CASP5). The FM generated models within 6 Å backbone rmsd for fragments of about 60–70 residues of a-helical proteins. For a CASP5 target that turned out to be natively unfolded, the trajectory obtained for this sequence uniquely failed to converge. Also, a new measure to evaluate structure predictions is presented and used along the standard CASP assessment methods. Finally, recent improvements in the prediction of β-sheet structures are briefly described.     

Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution

Recombinant Desulfovibrio vulgaris flavodoxin was produced inEscherichia coli. A complete backbone NMR assignment for the two-electronreduced protein revealed significant changes of chemical shift valuescompared to the oxidized protein, in particular for the flavinemononucleotide (FMN)-binding site. A comparison of homo- and heteronuclearNOESY spectra for the two redox states led to the assumption that reductionis not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgarisflavodoxin were investigated using two-dimensional¹⁵N-¹H correlation NMR spectroscopy.T₁, T₂ and NOE data are obtained for 95%of the backbone amide groups in both redox states. These values wereanalysed in terms of the model-free approach introduced by Lipari andSzabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. Acomparison of the two redox states indicates that in the reduced speciessignificantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H groupof FMN bound to the reduced protein compared to the oxidized state.     

1H and 15N resonance assignments and solution secondary structure of oxidized Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing ¹H–¹⁵N heteronuclear three-dimensional NMR experiments on uniformly ¹⁵N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with ³J_NH coupling constants and ¹H_N exchange data, indicate that the secondary structure consists of a five-stranded parallel -sheet and four -helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into ²H₂O, the ¹H^N3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.     

Native-specific stabilization of flavodoxin by the FMN cofactor: structural and thermodynamical explanation

Flavodoxins are useful models to investigate protein/cofactor interactions. The binding energy of the apoflavodoxin-FMN complex is high and therefore the holoflavodoxin is expected to be more stable than the apoprotein. This expectation has been challenged by reports on the stability of Desulfovibrio desulfuricans flavodoxin indicating that FMN binds to the unfolded polypeptide with similar affinity as to the native state, thus causing no net effect on protein stability. In previous work, we have analyzed in detail the stability of the apoflavodoxin from Anabaena PCC 7119 and the energetics of its functional complex with FMN. Here, we use the Anabaena holoprotein to directly investigate the contribution of the bound cofactor to protein stability through a detailed analysis of the chemical and thermal denaturation equilibria. Our data clearly shows that FMN binding largely stabilizes the protein towards both chemical and thermal denaturation, and that the stabilization observed at 25 degrees C in low ionic strength conditions is precisely the one expected if full release of the cofactor takes place upon flavodoxin unfolding. On the other hand, the binding of FMN to the native polypeptide is shown to simplify the thermal unfolding so that, while apoflavodoxin follows a three-state mechanism, the holoprotein unfolds in a two-state fashion. Comparison of the X-ray structure of native apoflavodoxin with the phi-structure of the thermal intermediate indicates that the increase in cooperativity driven by the cofactor originates in its preferential binding to the native state, which is a consequence of the disorganization in the intermediate of the FMN binding loops and of an adjacent longer loop.     

The Effect of Electrostatics on the Marginal Cooperativity of an Ultrafast Folding Protein

Proteins fold up by coordinating the different segments of their polypeptide chain through a network of weak cooperative interactions. Such cooperativity results in unfolding curves that are typically sigmoidal. However, we still do not know what factors modulate folding cooperativity or the minimal amount that ensures folding into specific three-dimensional structures. Here, we address these issues on BBL, a small helical protein that folds in microseconds via a marginally cooperative downhill process (Li, P., Oliva, F. Y., Naganathan, A. N., and Muñoz, V. (2009) Proc. Natl. Acad. Sci. USA. 106, 103–108). Particularly, we explore the effects of salt-induced screening of the electrostatic interactions in BBL at neutral pH and in acid-denatured BBL. Our results show that electrostatic screening stabilizes the native state of the neutral and protonated forms, inducing complete refolding of acid-denatured BBL. Furthermore, without net electrostatic interactions, the unfolding process becomes much less cooperative, as judged by the broadness of the equilibrium unfolding curve and the relaxation rate. Our experiments show that the marginally cooperative unfolding of BBL can still be made twice as broad while the protein retains its ability to fold into the native three-dimensional structure in microseconds. This result demonstrates experimentally that efficient folding does not require cooperativity, confirming predictions from theory and computer simulations and challenging the conventional biochemical paradigm. Furthermore, we conclude that electrostatic interactions are an important factor in determining folding cooperativity. Thus, electrostatic modulation by pH-salt and/or mutagenesis of charged residues emerges as an attractive tool for tuning folding cooperativity.     

Predicting protein folding cores by empirical potential functions

Theoretical and in vitro experiments suggest that protein folding cores form early in the process of folding, and that proteins may have evolved to optimize both folding speed and native-state stability. In our previous work (Chen et al., Structure, 14 (2006) 1401), we developed a set of empirical potential functions and used them to analyze interaction energies among secondary-structure elements in two β-sandwich proteins. Our work on this group of proteins demonstrated that the predicted folding core also harbors residues that form native-like interactions early in the folding reaction. In the current work, we have tested our empirical potential functions on structurally-different proteins for which the folding cores have been revealed by protein hydrogen-deuterium exchange experiments. Using a set of 29 unrelated proteins, which have been extensively studied in the literature, we demonstrate that the average prediction result from our method is significantly better than predictions based on other computational methods. Our study is an important step towards the ultimate goal of understanding the correlation between folding cores and native structures.     

Identical RNA-Protein Interactions in Vivo and in Vitro and a Scheme of Folding the Newly Synthesized Proteins by Ribosomes

A distinct three-dimensional shape of rRNA inside the ribosome is required for the peptidyl transfer activity of its peptidyltransferase center (PTC). In contrast, even the in vitro transcribed PTC RNA interacts with unfolded protein(s) at about five sites to let them attain their native states. We found that the same set of conserved nucleotides in the PTC interact identically with nascent and chemically unfolded proteins in vivo and in vitro, respectively. The time course of this interaction, difficult to follow in vivo, was observed in vitro. It suggested nucleation of folding of cytosolic globular proteins vectorially from hydrophilic N to hydrophobic C termini, consistent with our discovery of a regular arrangement of cumulative hydrophobic indices of the peptide segments of cytosolic proteins from N to C termini. Based on this observation, we propose a model here for the nucleation of folding of the nascent protein chain by the PTC.     

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins.     

Time-resolved fluorescence studies of flavodoxin. Fluorescence decay and fluorescence anisotropy decay of tryptophan in Desulfovibrio flavodoxins

The time-resolved fluorescence characteristics of tryptophan in flavodoxin isolated from the sulfate-reducing bacteria Desulfovibrio vulgaris and Desulfovibrio gigas have been examined. By comparing the results of protein preparations of normal and FMN-depleted flavodoxin, radiationless energy transfer from tryptophan to FMN has been demonstrated. Since the crystal structure of the D. vulgaris flavodoxin is known, transfer rate constants from the two excited states ¹ L _a and ¹ L _b can be calculated for both tryptophan residues (Trp 60 and Trp 140). Residue Trp 60, which is very close to the flavin, transfers energy very rapidly to FMN, whereas the rate of energy transfer from the remote Trp 140 to FMN is much smaller. Both tryptophan residues have the indole rings oriented in such a way that transfer will preferentially take place from the ¹ L _a excited state. The fluorescence decay of all protein preparations turned out to be complex, the parameter values being dependent on the emission wavelength. Several decay curves were analyzed globally using a model in which tryptophan is involved in some nanosecond relaxation process. A relaxation time of about 2 ns was found for both D. gigas apo- and holoflavodoxin. The fluorescence anisotropy decay of both Desulfovibrio FMN-depleted flavodoxins is exponential, whereas that of the two holoproteins is clearly non-exponential. The anisotropy decay was analyzed using the same model as that applied for fluorescence decay. The tryptophan residues turned out to be immobilized in the protein. A time constant of a few nanoseconds results from energy transfer from tryptophan to flavin, at least for D. gigas flavodoxin. The single tryptophan residue in D. gigas flavodoxin occupies a position in the polypeptide chain remote from the flavin prosthetic group. Because of the close resemblance of steady-state and time-resolved fluorescence properties of tryptophan in both flavodoxins, the center to center distance between tryptophan and FMN in D. gigas flavodoxin is probably very similar to the distance between Trp 140 and FMN in D. vulgaris flavodoxin (i.e. 20 Å). Offprint requests to: A.J.W.G. Visser     

Understanding the Mechanism of Prosegment-catalyzed Folding by Solution NMR Spectroscopy

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.     

Role of cations in stability of acidic protein Desulfovibrio desulfuricans apoflavodoxin

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)). A linear correlation of stabilizing effects with cation hydration properties suggests an important role of dehydration in efficient cation interaction with the protein. The effects on stability are due to preferential binding of one cation to native apoflavodoxin and results in an increase in thermal midpoint of 20 degrees C and the free energy of unfolding (at 20 degrees C) increases fivefold. Tuning of biophysical properties (such as folding and ligand/cofactor binding) of acidic proteins by cation binding may be important in vivo.