Folding mechanism of FIS, the intertwined, dimeric factor for inversion stimulation

FIS, the factor for inversion stimulation, from Escherichia coli and other enteric bacteria, is an interwined alpha-helical homodimer. Size exclusion chromatography and static light scattering measurements demonstrated that FIS is predominately a stable dimer at the concentrations (1-10 microM monomer) and buffer conditions employed in this study. The folding and unfolding of FIS were studied with both equilibrium and kinetic methods by circular dichroism using urea and guanidinium chloride (GdmCl) as the perturbants. The equilibrium folding is reversible and well-described by a two-state folding model, with stabilities at 10 degrees C of 15.2 kcal mol(-1) in urea and 13.5 kcal mol(-1) in GdmCl. The kinetic data are consistent with a two-step folding reaction where the two unfolded monomers associate to a dimeric intermediate within the mixing time for the stopped-flow instrument (<5 ms), and a slower, subsequent folding of the dimeric intermediate to the native dimer. Fits of the burst phase amplitudes as a function of denaturant showed that the free energy for the formation of the dimeric intermediate constitutes the majority of the stability of the folding (9.6 kcal mol(-1) in urea and 10.5 kcal mol(-1) in GdmCl). Folding-to-unfolding double jump kinetic experiments were also performed to monitor the formation of native dimer as a function of folding delay times. The data here demonstrate that the dimeric intermediate is obligatory and on-pathway. The folding mechanism of FIS, when compared to other intertwined, alpha-helical, homodimers, suggests that a transient kinetic dimeric intermediate may be a common feature of the folding of intertwined, segment-swapped, alpha-helical dimers.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

Spatial structure of dimeric a genetically engineered variant of green fluorescent protein EGFP-K162Q in the P61 crystal space group

The spatial structure of dimeric green fluorescent protein EGFP-K162Q with MDELYK (EGFPv) C-terminal deletion has been assigned in the P6₁ space group with resolution 1.34 Å by X-ray diffraction analysis. The results have been compared with X-ray diffraction data of monomeric EGFP (green biomarker with enhanced photophysical properties) assigned in another crystal space group, P2₁2₁2₁, with resolution 1.50 and 1.35 Å. Subunits in the EGFPv dimeric structure are located at 75° angle with the contact area ～800 Ų. The dimeric framework is stabilized by the six hydrogen bonds and central hydrophobic core of six residues. The root-mean-square deviation value for C^α atoms in 3–230 residues of the P6₁ and P2₁2₁2₁ crystal structures is 0.55 Å. The differential characteristics of EGFPv-P6₁ structure, compared to that of P2₁2₁2₁, is a noticeably different orientation of the Glu222 side chain, and a new conformation of the 155–159 loop fragment, characterized by deviations among the C^α atoms of superimposed structures reaching 4.6 Å for Lys156 and 5.5 Å for Lys158.     

Crystal structure of highly thermostable glycerol kinase from a hyperthermophilic archaeon in a dimeric form

The crystal structure of glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-GK) in a dimeric form was determined at a resolution of 2.4 A. This is the first crystal structure of a hyperthermophilic glycerol kinase. The overall structure of the Tk-GK dimer is very similar to that of the Escherichia coli glycerol kinase (Ec-GK) dimer. However, two dimers of Ec-GK can associate into a tetramer with a twofold axis, whereas those of Tk-GK cannot. This may be the reason why Tk-GK is not inhibited by fructose 1,6-bisphosphate, because the fructose 1,6-bisphosphate binding site is produced only when a tetrameric structure is formed. Differential scanning calorimetry analyses indicate that Tk-GK is a highly thermostable protein with a melting temperature (T(m)) of 105.4 degrees C for the major transition. This value is higher than that of Ec-GK by 34.1 degrees C. Comparison of the crystal structures of Tk-GK and Ec-GK indicate that there is a marked difference in the number of ion pairs in the alpha16 helix. Four ion pairs, termed IP1-IP4, are formed in this helix in the Tk-GK structure. To examine whether these ion pairs contribute to the stabilization of Tk-GK, four Tk-GK and four Ec-GK derivatives with reciprocal mutations at the IP1-IP4 sites were constructed. The determination of their stabilities indicates that the removal of each ion pair does not affect the stability of Tk-GK significantly, whereas the mutations designed to introduce one of these ion pairs stabilize or destabilize Ec-GK considerably. These results suggest that the ion pairs in the alpha16 helix contribute to the stabilization of Tk-GK in a cooperative manner.     

A pH-regulated dimeric bouquet in the structure of von Willebrand factor

At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal ～1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.     

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.     

A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(+/-0.25) A and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2. 2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(+/-0.43) A, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) A, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23. 5 A). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.     

Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation

The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins.     

Thermal unfolding pathway for the thermostable P22 tailspike endorhamnosidase

The conditions in which protein stability is biologically or industrially relevant frequently differ from those in which reversible denaturation is studied. The trimeric tailspike endorhamnosidase of phage P22 is a viral structural protein which exhibits high stability to heat, proteases, and detergents under a range of environmental conditions. Its intracellular folding pathway includes monomeric and trimeric folding intermediates and has been the subject of detailed genetic analysis. To understand the basis of tailspike thermostability, we have examined the kinetics of thermal and detergent unfolding. During thermal unfolding of the tailspike, a metastable unfolding intermediate accumulates which can be trapped in the cold or in the presence of SDS. This species is still trimeric, but has lost the ability to bind to virus capsids and, unlike the native trimer, is partially susceptible to protease digestion. Its N-terminal regions, containing about 110 residues, are unfolded whereas the central regions and the C-termini of the polypeptide chains are still in the folded state. Thus, the initiation step in thermal denaturation is the unfolding of the N-termini, but melting of the intermediate represents a second kinetic barrier in the denaturation process. This two-step unfolding is unusually slow at elevated temperature; for instance, in 2% SDS at 65 degrees C, the unfolding rate constant is 1.1 x 10(-3) s-1 for the transition from the native to the unfolding intermediate and 4.0 x 10(-5) s-1 for the transition from the intermediate to the unfolded chains. The sequential unfolding pathway explains the insensitivity of the apparent Tm to the presence of temperature-sensitive folding mutations [Sturtevant, J. M., Yu, M.-H., Haase-Pettingell, C., & King, J. (1989) J. Biol. Chem. 264, 10693-10698] which are located in the central region of the chain. The metastable unfolding intermediate has not been detected in the forward folding pathway occurring at lower temperatures. The early stage of the high-temperature thermal unfolding pathway is not the reverse of the late stage of the low-temperature folding pathway.     

Stabilization of the intermediate in frameshift mutation

A mismatch repair, proofreading deficient mutant of Escherichia coli lost a C from a C8 run at a rate 10 times higher than the loss of A from an A8 sequence in the same double mutant. This greater frameshift instability of a homopolymeric run of C's may be due to stabilization of a stacked intermediate. Gain of a (CA) unit in a similarly constructed (CA)15 sequence occurred at a rate about 1/3 that previously reported for a (CA)14 construct losing a (CA) repeat unit.     

A truncated peptide model of the mutant P61A FIS forms a stable dimer

Factor for inversion stimulation (FIS) is a 98-residue homodimeric DNA-binding protein involved in several different cellular processes including DNA inversion and the regulation of multiple genes. FIS contains a flexible and functionally important N-terminus followed by four helices (A-D), the last two of which consist of the DNA-binding region. Helix B, which comprises the main dimerization interface has a 20 degrees kink at its center that was originally thought to be caused by the presence of a proline at position 61. However, it was later shown that the kink remained largely intact and that FIS retained its native-like function when the proline was mutated to an alanine. We previously showed that the P61A mutation increased the stability of FIS, but decreased its equilibrium denaturation cooperativity apparently due to preferential stabilization of the B-helix. Here we studied a peptide of P61A FIS, corresponding to residues 26-71 (26-71(A3) FIS), which encompasses the dimer interface (helices A and B). Circular dichroism (CD) and size-exclusion chromatography/multi-angle light scattering showed that the peptide was alpha-helical and dimeric, respectively, as expected based on the 3D structure of FIS. Urea-induced equilibrium denaturation experiments monitored by far-UV CD revealed a concentration-dependent transition, and data analysis based on a N2<-->2U model yielded a DeltaG of approximately -10 kcal/mol. Our results suggest that 26-71(A3) FIS can form a stable dimeric structure despite lacking the N- and C-terminus of native FIS.     

Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure

The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUalpha(2) and EcHUbeta(2), and a heterodimer, EcHUalphabeta. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E.coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N(2)<-->2D), this present thermodynamic study shows that the three E.coli dimers melt according to a two-step mechanism (N(2)<-->I(2)<-->2D). The native dimer, N(2), melts partially into a dimeric intermediate, I(2), which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUalpha(2) dimer has been solved. Comparative thermodynamic and structural analysis between EcHUalpha(2) and the HU homodimer from Bacillus stearothermophilus suggests that the E.coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I(2), corresponds to an HU dimer having partly lost its alpha-helices. The partially unfolded dimer I(2) is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N(2)<-->I(2) equilibrium plays a central role in the physiology of E.coli HU. The I(2) molecular species seems to be the EcHUbeta(2) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation. Besides, I(2) might be required in E.coli for the HU chain exchange, which allows the heterodimer formation from homodimers.     

A phaseolus mutation results in a reduced level of lectin mRNA

Summary The molecular basis of a lectin gene mutation in the Phaseolus vulgaris cultivar Pinto was investigated. Rocket immunoelectrophoresis studies showed that seed lectin is reduced approximately 40-fold in comparison to the normal Phaseolus vulgaris cultivar Contender. DNA gel blot studies using a lectin cDNA probe suggested that the Pinto and Contender varieties contain similar numbers of lectin genes, although qualitative differences were observed in the gel blot banding patterns. Hybridization of the lectin cDNA probe to gel blots containing normal and mutant embryo mRNAs produced a 1 kb mRNA band in both mRNA populations. However, the amount of lectin mRNA was reduced approximately tenfold in the mutant Pinto cultivar. Together, these findings suggest that the reduced seed lectin level is due, in part, to a reduction in seed lectin mRNA.Abbreviations mRNA messenger RNA - cDNA complementary DNA - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - BSA bovine serum albumin - kd kilodaltons - kb kilobases     

Evidence for a monomeric intermediate in the reversible unfolding of F factor TraM

F factor TraM is essential for efficient bacterial conjugation, but its molecular function is not clear. Because the physical properties of TraM may provide clues to its role in conjugation, we have characterized the TraM oligomerization equilibrium. We show that the reversible unfolding transition is non-two-state, indicating the presence of at least one intermediate. Analytical ultracentrifugation experiments indicate that the first phase of unfolding involves dissociation of the tetramer into folded monomers, which are subsequently unfolded to the denatured state in the second phase. Furthermore, we show that a C-terminal domain isolated by limited proteolysis is tetrameric in solution, like the full-length protein, and that its loss of structure correlates with dissociation of the TraM tetramer. Unfolding of the individual domains indicates that the N- and C-terminal regions act cooperatively to stabilize the full-length protein. Together, these experiments suggest structural overlap of regions important for oligomerization and DNA binding. We propose that modulating the oligomerization equilibrium of TraM may regulate its essential activity in bacterial conjugation.