Transmembrane helix-helix association: relative stabilities at low pH

We have previously studied the unfolding equilibrium of bacterioopsin in a single phase solvent, using F?rster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterioopsin perturbed by increasing ethanol concentrations [Nannepaga et al. (2004) Biochemistry 43, 50-59]. We have further investigated this transition and find that the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterioopsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are 5.46 for residues 72-248 (Cys 163) and 7.36 for residues 1-166 (Lys 41). When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding in the range of -0.54 to -3.5 kcal/mol. The results suggest that the C-terminal helix of bacterioopsin is less stably folded than the N-terminal helices. We analyzed the pairwise helix-helix interaction surfaces of bacteriorhodopsin and three other seven-transmembrane-helix proteins on the basis of crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl-terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high-resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helix-helix interactions.     

Structural stability of the erythrocyte anion transporter, band 3, in native membranes and in detergent micelles.

The exothermic thermal denaturation transition of band 3, the anion transporter of the human erythrocyte membranes, has been studied by differential scanning calorimetry, in ghost membranes and in nonionic detergent micelles. In detergent micelles the transmembrane domain of band 3 gave an irreversible denaturation transition (C transition). However, no thermal transition was observed for the N-terminal cytoplasmic domain when band 3 was solubilised in detergent micelles. A reduction in enthalpy (190-300 kcal mol-1) with an accompanying decrease in thermal denaturation temperatures (48-60 degrees C) for the C transition was observed in detergent solubilised band 3 when compared with ghost membranes. Unlike ghost membranes, two thermal transitions for band 3 in detergent micelles were observed for the C transition when in the presence of excess covalent inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), which derive from the thermal unfolding of a single protein with two different thermal stabilities; DIDS-stabilised (75 degrees C) and DIDS-insensitive (62 degrees C). A reduction in the denaturation temperature for the transmembrane domain of band 3 was observed when compared with intact band 3 although no significant differences was observed in the corresponding enthalpy values. This indicates some cooperativity of the two domains of band 3 in maintaining the transmembrane conformation. The results presented in this study show that detergents of intermediate micelle size (e.g. Triton X-100 and C12E8) are required for optimal thermal stability of band 3.     

Structure and dynamics at N- and C-terminal regions of intrinsically disordered human c-Myc PEST degron reveal a pH-induced transition

We investigated the structure and Brownian rotational motion of the PEST region (201-268) from human c-Myc oncoprotein, whose overexpression/dysregulation is associated with various types of cancer. The 77-residue PEST fragment revealed a large Stokes radius (~3.1 nm) and CD spectrum highlighting abundance of disordered structure. Changes in structure/dynamics at two specific sites in PEST degron were observed using time-resolved fluorescence spectroscopy by labeling Cys⁹ near N-terminal with dansyl probe and inserting a Trp⁷⁰ near C-terminal (PEST M1). Trp in PEST M1 at pH 3 was inaccessible to quencher, showed hindered segmental motion and slow global rotation (~30 ns) in contrast to N-terminal where the dansyl probe was free, exposed with fast global rotation (~5 ns). Remarkably, this large monomeric structure at acidic pH was retained irrespective of ionic strength (0.03-0.25 M) and partially so in presence of 6 M Gdn.HCl. With gradual increase in pH, a structural transition (~pH 4.8) into a more exposed and freely rotating Trp was noticeable. Interestingly, the induced structure at C-terminal also influenced the dynamics of dansyl probe near N-terminal, which otherwise remained unstructured at pH > 5. FRET measurements confirmed a 11 Å decrease in distance between dansyl and indole at pH 4 compared to pH 9, coinciding with enhanced ANS binding and increase in strand/helix population in both PEST fragments. The protonation of glutamate/aspartate residues in C-terminal region of PEST is implicated in this disorder-order transition. This may have a bearing on the role of PEST in endocytic trafficking of eukaryotic proteins.     

Investigation of the structural stability of hUBF HMG box 5 by native-state hydrogen exchange

HMG box 5 of human upstream binding factor (hUBF) consists of three alpha-helices arranged in an L-shape with a hydrophobic core embraced by these helices and stabilized by extensive hydrophobic interactions between nonpolar residues around the core. The GdmCl-induced equilibrium unfolding transition of HMG box 5 of hUBF was monitored by both circular dichroism (CD) and fluorescence spectra. A cooperative two-state unfolding process was observed. The unfolding free energy, DeltaGU(D2O), and the cooperativity of the unfolding reaction, m, are 4.6 +/- 0.16 kcal x mol-1 and 1.62 +/- 0.06 kcal x mol-1 x M-1, respectively. Native-state hydrogen exchange (NHX) experiments under EX2 conditions were performed. NHX results clearly show that the hydrophobic core among the three helices is a slow-exchange core. The three helices would not contribute equally to the stability of the native protein. Helix 3 appears to contribute the least to the stability. The NHX data have also allowed the local, subglobal, and global unfolding structures of hUBF HMG box 5 to be dissected, and common global and subglobal unfolding units were successfully detected.     

Protein self-assembly and lipid binding in the folding of the potassium channel KcsA

Moderate concentrations of the alcohol 2,2,2-trifluoroethanol (TFE) cause the coupled unfolding and dissociation into subunits of the homotetrameric potassium channel KcsA, in a process that is partially irreversible when the protein is solubilized in plain dodecyl beta-d-maltoside (DDM) micelles [Barrera et al. (2005) Biochemistry 44, 14344-52]. Here we report that the transition from the folded tetramer to the unfolded monomer becomes completely reversible when KcsA is solubilized in mixed micelles composed of the detergent DDM and the lipids DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]). This result suggests that lipids may act as effectors in the tetramerization of KcsA. The observed reversibility allowed the determination of the standard free energy of the folding reaction of KcsA: DeltaG = 30.5 +/- 3.1 kcal x mol-1. We also observed that, prior to the unfolding of the tetramer, the presence of lower TFE concentrations causes the disassembly of supramolecular clusters of KcsA into the individual tetrameric molecules. Within the limits of experimental resolution, this is also a reversible process, but unlike the tetramer to monomer transition from above, the level of clustering is not influenced by the presence of solubilized lipids. These observations suggest a distinct role of the lipids in the different in vitro assembly steps (folding/tetramerization and clustering) of KcsA.     

Direct sugar binding to LacY measured by resonance energy transfer

Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for F?rster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the F?rster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.     

MD simulations of Mistic: conformational stability in detergent micelles and water

Mistic is an unusual membrane protein from Bacillus subtilis. It appears to fold and insert autonomously into a lipid bilayer and has been suggested as a tool that aids the targeting of eukaryotic membrane proteins to bacterial membranes. The NMR structure of Mistic in detergent (LDAO) micelles has revealed it to be a four alpha-helix bundle. From a structural perspective, Mistic does not resemble other membrane proteins. Its external surface is not very hydrophobic, and standard methods do not predict any of its helices to be in the transmembrane orientation. Molecular dynamics simulations (simulation times approximately 30 ns) in water and in detergent micelles have been used to explore the conformational stability of Mistic as a function of its environment. In water, the protein is stable, exhibiting no significant change in fold on a 30 ns time scale. In contrast, in three simulations in detergent micelles, the partial unfolding of Mistic occurred, whereby the H4 helix drifted away from the H1-H3 core. This was due to the penetration of detergent molecules between H4 and the remainder of the protein. This is unlike the behavior of several other membrane proteins, both alpha-helix bundles and beta-barrels, in comparable detergent micelle simulations. The unfolding of H4 from the H1-H3 core of Mistic could be partially reversed by a simulation in which the detergent molecules were removed, and the unfolded protein was simulated in water. These results suggest that Mistic may not be a stable integrated membrane protein but rather that it may undergo a conformational change upon interaction with a membrane or membrane-like environment.     

Sequential unfolding of individual helices of bacterioopsin observed in molecular dynamics simulations of extraction from the purple membrane

Multiple molecular dynamics simulations of bacterioopsin pulling from its C-terminus show that its alpha-helices unfold individually. In the first metastable state observed in the simulations, helix G is unfolded at its C-terminal segment while the rest of helix G (residues 200-216) is folded and opposes resistance because of a salt-bridge network consisting of Asp-212 and Lys-216 on helix G and Arg-82 and Asp-85 on helix C. Helix G unfolds inside the bundle because the external force is applied to its C-terminal end in a direction perpendicular to the surface of the membrane. Inversely, helix F has to flip by 180 degrees to exit from the membrane because the applied force and the helical N-C axis point in opposite directions. At the highest peak of the force, which cannot be interpreted in single-molecule force spectroscopy experiments, helix F has a pronounced kink at Pro-186. Mutation of Pro-186 and/or the charged side chains mentioned above, which are involved in very favorable electrostatic interactions in the low-dielectric region of the membrane, are expected to reduce the highest peak of the force. Helices E and D unfold in a similar way to helices G and F, respectively. Hence, the force-distance profile and sequence of events during forced unfolding of bacterioopsin are influenced by the up-and-down topology of the seven-helix bundle. The sequential extraction of individual helices from the membrane suggests that the spontaneous (un)folding of bacterioopsin proceeds through metastable bundles of fewer than seven helices. The metastable states observed in the simulations provide atomic level evidence that corroborates the interpretation of very recent force spectroscopy experiments of bacteriorhodopsin refolding.     

Relative Domain Folding and Stability of a Membrane Transport Protein

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately + 2 kcal mol^− 1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of + 2 kcal mol^− 1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains.     

Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar

The denaturant-induced unfolding kinetics of the 89-residue protein, barstar, have been examined using fluorescence resonance energy transfer (FRET) at 25 degrees C and pH 8.0. The core tryptophan, Trp53, in barstar serves as a fluorescence donor, and a thionitrobenzoic acid moiety (TNB) attached to a cysteine residue acts as an acceptor to form an efficient FRET pair. Four different single-cysteine containing mutants of barstar with cysteine residues at positions 25, 40, 62, and 82 were studied. The unfolding kinetics of the four mutant forms of barstar were monitored by measurement of the changes in the fluorescence intensity of Trp53 in the unlabeled and TNB-labeled proteins. The rate of change of fluorescence of the single-tryptophan residue, Trp53, in the unlabeled protein, where no FRET occurs, yields the rate of solvation of the core. This rate is similar for all four unlabeled proteins. The rate of the increase in the fluorescence of Trp53 in the labeled protein, where FRET from the tryptophan to the TNB label occurs, yields the rate of decrease in FRET efficiency during unfolding. The decrease in FRET efficiency for proteins labeled at either of the two buried positions (Cys40 or Cys82) occurs at a rate similar to the rate of core solvation. The decrease in FRET efficiency for the acceptor at Cys40 is also shown to be sensitive to the isomerization of the Tyr47-Pro48 cis bond. For the proteins where the label is at a solvent-exposed position (Cys25 and Cys62), the decrease in FRET efficiency occurs in two kinetic phases; 15-25% of the FRET efficiency decreases in the faster phase, and the remaining FRET efficiency decreases in a slower phase, the rate of which is the same as the rate of core solvation. These results clearly indicate that, during unfolding, the protein surface expands faster than, and independently of, water intrusion into the core.     

Snapshots of protein folding. A study on the multiple transition state pathway of cytochrome c(551) from Pseudomonas aeruginosa

Cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa is a small protein (82 residues) that folds via a three-state pathway with the accumulation in the microsecond time-range of a compact collapsed intermediate. The presence of a single His residue, at position 16, permits the study of the refolding at pH 7.0 in the absence of miscoordination events. Here, we report on folding kinetics in the millisecond time-range as a function of urea under different pH conditions. Analysis of this process (over-and-above proline cis-trans isomerization) at pH 7.0, suggests the existence of a multiple transition state pathway in which we postulate three transition states. Taking advantage of site-directed mutagenesis we propose that the first "unfolded-like" transition state (t(1)) originates from the electrostatic properties of the collapsed state, while the second transition state (t(2)) involves the interaction between the N and C-terminal helices and is stabilized by the salt bridge between Lys10 and Glu70 ( approximately 1 kcal mol(-1)). Our results suggest that, contrary to other cytochromes c, the roll-over effect observed for cyt c(551) at low denaturant concentration can be interpreted in terms of a broad energy barrier without population of any intermediates. The third and more "native-like" transition state (M) can be associated with the breaking/formation of the Fe(3+)-Met61 bond. This strong interaction is stabilized by the hydrogen bond between Trp56 and heme propionate 17 (HP-17) as suggested by the increase in the unfolding rate at high denaturant concentration of the Trp56Phe site-directed mutant.     

Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly.

The M2 protein from influenza A virus is a 97-residue homotetrameric membrane protein that functions as a proton channel. To determine the features required for the assembly of this protein into its native tetrameric state, the protein was prepared by total synthesis using native chemical ligation of unprotected peptide segments. Circular dichroism spectroscopy of synthetic M2 protein in dodecylphosphocholine (DPC) micelles indicated that approximately 40 residues were in an alpha-helical secondary structure. The tetramerization of the full-length protein was compared to that of a 25-residue transmembrane (TM) fragment. Analytical ultracentrifugation demonstrated that both the peptide and the full-length protein in DPC micelles existed in a monomer-tetramer equilibrium. Comparison of the association constants for the two sequences showed the free energy of tetramerization of the full-length protein was more favorable by approximately 7 kcal/mol. Partial proteolysis of DPC-solubilized M2 was used as a further probe of the structure of the full-length protein. A 15-20-residue segment C-terminal to the membrane-spanning region was found to be highly resistant to digestion by chymotrypsin and trypsin. This region, which we have modeled as an extension of the TM helices, may help to stabilize the tetrameric assembly.     

Partial unfolding of dodecameric glutamine synthetase from Escherichia coli: temperature-induced, reversible transitions of two domains

Glutamine synthetase (GS), Mr 622,000, from Escherichia coli contains 12 active sites formed at heterologous interfaces between subunits [Almassy, R. J., Janson, C. A., Hamlin, R., Xuong, N.-H., & Eisenberg, D. (1986) Nature (London) 323, 304-309]. Temperature-induced changes in UV spectra from 3 to 68 degrees C were reversible with the Mn2+- or Mg2+-enzyme at pH 7.0 (50 degrees C) in 100 mM KCl. No dissociation or aggregation of dodecamer occurred at high temperatures. The thermal transition involves the exposure of approximately 0.7 of the 2 Trp residues/subunit (by UV difference spectroscopy) and 2 of the 17 Tyr residues/subunit (change in exposure from 4.7 to 6.7 Tyr/subunit by second-derivative spectral analysis). Monitoring changes in Trp and Tyr exposure independently gives data that conform to a two-state model for partial unfolding with Tm values (where delta G unfolding = 0) differing by 2-3 degrees C at each level of [Mn2+] studied and with average delta HvH values of 80 and 94 kcal/mol, respectively. These observations suggest that two regions of the oligomeric structure unfold separately as independent transitions (random model). However, the data can be fit equally with a sequential model in which the Trp transition occurs first upon heating. By fitting with either model, Tm values increase from approximately 47 to approximately 54 degrees C with increasing free [Mn2+] from 3.6 to 49 microM but decrease from approximately 54 to approximately 43 degrees C by further increasing free [Mn2+] from 0.05 to 10 mM; such behavior indicates that the high-temperature form of the enzyme binds Mn2+ more weakly but has more binding sites than the native enzyme. The high-temperature Mn-enzyme form is somewhat less unfolded than is the catalytically inactive apoenzyme, which undergoes no further Trp or Tyr exposure on heating and therefore is assumed to be the high-temperature form of divalent cation-free GS. Adding substrates [ADP, L-Met-(SR)-sulfoximine, Gln, Gln + NH2OH, or Gln + ADP] to Mn.GS increased Tm to varying extents by preferential binding to the folded form. Indeed, the transition-state analogue complex GS.(Mn2.ADP.L-Met-(S)-sulfoximine phosphate)12 was stable in the folded form to at least 72 degrees C. Moreover, an Arrhenius plot for gamma-glutamyl transfer activity was linear from 4 to 72 degrees C with Ea = 18.3 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein unfolding in detergents: effect of micelle structure,ionic strength,pH, and temperature

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents.     

Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition

The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.     

Thermodynamic investigations of cytochrome b5 unfolding. II. Detergent-solubilized cytochrome b5 in solution and in a reconstituted system with dimyristoyl phosphatidylcholine

Thermal unfolding of the detergent-solubilized cytochrome b5 was investigated by scanning calorimetry. The protein shows different thermostability in the presence and absence of detergent, and it achieves the maximal transition temperature after incorporation into dimyristoyl phosphatidylcholine liposomes. However, transition temperature and Gibbs energy change at unfolding are still lower than that of the tryptic fragment of cytochrome b5 in aqueous solution. Cytochrome b5 undergoes in aqueous solution in the absence of detergent an irreversible, complicated transition, but it remains in the associated state after thermal denaturation. Half transition temperature, enthalpy and heat capacity changes of cytochrome b5 unfolding under various external conditions are reported and compared with the corresponding values of the tryptic fragment of the protein. The thermodynamic data and independent results are suitable for detailing a model proposed by Tanford (The Hydrophobic Effect (1980), pp. 205-211, John Wiley & Sons, New York) for the spatial arrangement of the protein within the membrane.     

A theoretical DFT investigation of the lysozyme mechanism: computational evidence for a covalent intermediate pathway

To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and -118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and -104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima (Tm,Fl lambda) and those derived from DSC (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (Tm,Fl int) and from DSC (Tm,DSC), respectively, are quite similar, and thus, no local rearrangements are observed upon heat-induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl-induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped-flow fluorescence measurements. By phi-value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native.     

Direct observation of protein folding in nanoenvironments using a molecular ruler

We observe folding of horse heart cytochrome c in various environments including nano-compartments (micelles and reverse micelles). Using picosecond-resolved Förster resonance energy transfer (FRET) dynamics of an extrinsic covalently attached probe dansyl (donor) at the surface of the protein to a heme group (acceptor) embedded inside the protein, we measured angstrom-resolved donor–acceptor distances in the environments. The overall structural perturbations of the protein revealed from the FRET experiments are in close agreement with our circular dichroism (CD) and dynamic light scattering (DLS) studies on the protein in a variety of solution conditions. The change of segmental motion of the protein due to imposed restriction in the nano-compartments compared to that in bulk buffer is also revealed by temporal fluorescence anisotropy of the dansyl probe.     

Evidence for post-translational membrane insertion of the integral membrane protein bacterioopsin expressed in the heterologous halophilic archaeon Haloferax volcanii

The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.     

Equilibrium unfolding mechanism of chicken muscle triose phosphate isomerase

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as DeltaG degrees =3.54 kcal.mol(-1), and m(G) = 0.67 kcal.mol(-1)M(-1), which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, DeltaH(cal)/DeltaH(vH), was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, DeltaH(vH), was estimated as about 200 kcal.mol(-1). These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.