Dramatic structural and thermodynamic consequences of repacking a protein's hydrophobic core

BACKGROUND: Rop is an RNA binding, dimeric, four-helix bundle protein with a well-defined, regular hydrophobic core ideally suited for redesign studies. A family of Rop variants in which the hydrophobic core was systematically redesigned has previously been created and characterized. RESULTS: We present a structural and thermodynamic analysis of Ala2Ile2-6, a variant of Rop with an extensively redesigned hydrophobic core. The structure of Ala2Ile2-6 reveals a completely new fold formed by a conformational "flip" of the two protomers around the dimeric interface. The free-energy profile of Ala2Ile2-6 is also very different from that of wild-type Rop. Ala2Ile2-6 has a higher melting temperature than Rop, but undergoes a slightly smaller free-energy change on unfolding. CONCLUSIONS: The structure of Ala2Ile2-6, along with molecular modeling results, demonstrate the importance of tight packing of core residues and the adoption of favorable core side chain rotamer values in determining helix-helix interactions in the four-helix bundle fold. Structural disorder at the N and C termini of Ala2Ile2-6 provides a basis for the large differences in the enthalpy and entropy of Ala2Ile2-6 folding compared with wildtype Rop.     

Analysis of peptide design in four-, five-, and six-helix bundle template assembled synthetic protein molecules

Four-, five-, and six-helix bundle template assembled synthetic proteins (TASPs) have been synthesized using disulfide bonds between cavitand templates and peptides, and characterized in terms of stability and structural specificity. The peptide sequence (CGGGEELLKKLEE LLKKG) used was originally designed for a four-helix bundle. The TASPs were analyzed using CD spectroscopy, chemical denaturation studies, NMR spectroscopy, sedimentation equilibria studies, and hydrophobic dye binding studies to determine the effect of a single peptide sequence when incorporated into bundles with different numbers of helices. If the design was indeed idealized for a four-helix bundle, then the five- and six-helix bundles should be less stable and manifest lower conformational specificity. The TASPs all demonstrated high stability and cooperative unfolding. However, the four-helix bundle was found to be significantly more stable and nativelike compared to the five- and six-helix bundles. This suggests that the peptide sequence is specific to the four-helix bundle, as designed. This result demonstrates the ability to design de novo proteins with specified structure, not just generic stability.     

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.     

Investigating the functional role of a buried interchain aromatic cluster in Escherichia coli GrpE dimer

Aromatic clusters in the core of proteins are often involved in imparting structural stability to proteins. However, their functional importance is not always clear. In this study, we investigate the thermosensing role of a phenylalanine cluster present in the GrpE homodimer. GrpE, which acts as a nucleotide exchange factor for the molecular chaperone DnaK, is well known for its thermosensing activity resulting from temperature-dependent structural changes that allow control of chaperone function. Using mutational analysis, we show that an interchain phenylalanine cluster in a four-helix bundle of the GrpE homodimer assists in the thermosensing ability of the co-chaperone. Substitution of aromatic residues with hydrophobic ones in the core of the four-helix bundle reduces the thermal stability of the bundle and that of a connected coiled-coil domain, which impacts thermosensing. Cell growth assays and SEM images of the mutants show filamentous growth of Escherichia coli cells at 42°C, which corroborates with the defect in thermosensing. Our work suggests that the interchain edge-to-face aromatic cluster is important for the propagation of the structural signal from the coiled-coil domain to the four-helical bundle of GrpE, thus facilitating GrpE-mediated thermosensing in bacteria.     

Conformation specificity and arene binding in a peptide composed only of Lys, Ile, Ala and Gly

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I. Using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we find kinks in the helices of KIA7I and multiple C-terminal conformations. These results suggest that the C-terminal Ile residue does not completely occupy the hydrophobic pocket that is filled by aromatic side-chains in well-folded KIA7 variants. The capacity of arenes to fill this cavity was tested. Using NMR, we show that benzene and phenol can bind KIA7I, but do not bind the well-folded variant KIA7W or hen egg white lysozyme. Benzene also binds Aβ_1–40, a mostly disordered polypeptide implicated in Alzheimer’s disease. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence is further enhanced in the presence of both KIA7I and arenes relative to KIA7I alone. This ANS fluorescence enhancement is stronger for smaller and less polar arenes and less ordered KIA variants. These results suggest that arenes are not confined to the pocket, but penetrate and loosen the hydrophobic core of KIA7I.     

Synthesis and biophysical characterization of engineered topographic immunogenic determinants with alpha alpha topology.

Model peptides with predetermined secondary, tertiary, and quaternary conformation have been successfully designed, synthesized, and characterized in an attempt to mimic the three-dimensional structure of an antigenic determinant. This work is a continuing effort to map the antigenic structure of the protein antigen lactate dehydrogenase C4 (LDH-C4) to develop a contraceptive vaccine. A putative topographic determinant with alpha alpha topology which associates into four-helix bundles was designed on the basis of the framework model of protein folding. An idealized amphiphilic 18-residue sequence (alpha 1) and a 40-residue alpha alpha fold (alpha 3) have been shown to form stable 4-helix structures in solution with a free energy of association on the order of -20.8 kcal/mol (tetramerization of alpha 1) and -7.8 kcal/mol (dimerization of alpha 3). Both alpha 1 and alpha 3 form stable monolayers at the air-water interface. The CD spectra of Langmuir-Blodgett monolayers are characteristically alpha-helical. Both CD and FTIR spectroscopic studies reval a high degree of secondary structure. The SAXS data strongly suggest that the helices are arranged in a four-helix bundle since the radius of gyration of 17.2 A and the vector distribution function are indicative of a prolate ellipsoid of axial dimensions and molecular weight appropriate for the four-helix bundle. The major contribution to the formation and stabilization of alpha 1 and alpha 3 is believed to be hydrophobic interaction between the amphiphilic alpha-helices. The displayed heptad repeat, helix dipole, ion pairs, and the loop sequence may have also contributed to the overall stability and antiparallel packing of the helices. A detailed structural analysis of a relevant topographic immunogenic determinant will elucidate the nature of antigen-antibody interactions as well as provide insight into protein folding intermediates.     

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Stability of diether C(25,25) liposomes from the hyperthermophilic archaeon Aeropyrum pernix K1

Temperature and pH effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the Aeropyrum pernix. We determined the permeability of C_25,25 liposomes using fluorescence intensity of released calcein. At pH 7.0 and 9.0, and from 85 °C to 98 °C, only 10% of entrapped calcein was released. After 10 h at 90 °C, calcein release reached 27%, independent of pH. Fluorescence anisotropy measurements of hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene revealed gradual changes up to 60 °C. At higher temperatures, the anisotropy did not change significantly. Fluorescence alone did not provide detailed and direct structural information about these C_25,25 liposomes, so we used electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC). From EPR spectra, mean membrane fluidity determined according to maximal hyperfine splitting and empirical correlation times showed continuous increases with temperature. Computer simulation of EPR spectra showed heterogeneous membranes of these C_25,25 liposomes: at low temperatures, they showed three types of membrane regions characterized by different motional modes. Above 65 °C, the membrane becomes homogeneous with only one fluid-like region. DSC thermograms of C_25,25 liposomes reveal a very broad and endothermic transition in the temperature range from 0 °C to 40 °C.     

Fluidity properties of isolated chloroplast thylakoid lipids

Chloroplast thylakoid lipids have been isolated free of photosynthetic pigments using a combination of high performance liquid and thin layer chromatography. The hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into aqueous dispersions of the isolated lipids in order to investigate dynamic and structural properties of the resulting bilayer membranes. Time dependent fluorescence anisotropy decays have been measured and analysed assuming the wobbling-in-cone model (Kinosita et al., Biophys J 20 (1977) 289–305). The DPH fluorescence lifetimes and the static and dynamic fluorescence anisotropy decay parameters for the probe in a total lipid mixture or in pure digalactosyldiacylglycerol (DGDG), changed in a predictable way with increasing temperature (10°–36°C). For a given temperature, it was found that the total lipid mixture was in general less ordered and showed greater dynamic motion as judged from DPH fluorescence anisotropy and compared with the pure DGDG system, although at 36°C differences in dynamic parameters were less evident. Overall the results obtained emphasize the highly fluid nature of thylakoid membrane lipids and give a basis for investigating how intrinsic proteins modify structural and dynamic properties of the in vivo membrane.     

Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin-binding site (VBS), folds into a five-helix bundle whereas talin 656–789 is a four-helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five-helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.     

Lessons on protein structure from interleukin-4: All disulfides are not created equal

Interleukin-4 (IL-4) is a hematopoietic cytokine composed by a four-helix bundle stabilized by an antiparallel beta-sheet and three disulfide bonds: Cys3-Cys127, Cys24-Cys65, and Cys46-Cys99. IL-4 is involved in several immune responses associated to infection, allergy, autoimmunity, and cancer. Besides its physiological relevance, IL-4 is often used as a “model” for protein design and engineering. Hence, to understand the role of each disulfide in the structure and dynamics of IL-4, we carried out several spectroscopic analyses (circular dichroism [CD], fluorescence, nuclear magnetic resonance [NMR]), and molecular dynamics (MD) simulations on wild-type IL-4 and four IL-4 disulfide mutants. All disulfide mutants showed loss of structure, altered interhelical angles, and looser core packings, showing that all disulfides are relevant for maintaining the overall fold and stability of the four-helix bundle motif, even at very low pH. In the absence of the disulfide connecting both protein termini Cys3-Cys127, C3T-IL4 showed a less packed protein core, loss of secondary structure (~9%) and fast motions on the sub-nanosecond time scale (lower S² order parameters and larger τ_c correlation time), especially at the two protein termini, loops, beginning of helix A and end of helix D. In the absence of Cys24-Cys65, C24T-IL4 presented shorter alpha-helices (14% loss in helical content), altered interhelical angles, less propensity to form the small anti-parallel beta-sheet and increased dynamics. Simultaneously deprived of two disulfides (Cys3-Cys127 and Cys24-Cys65), IL-4 formed a partially folded “molten globule” with high 8-anilino-1-naphtalenesulphonic acid-binding affinity and considerable loss of secondary structure (~50%decrease), as shown by the far UV-CD, NMR, and MD data.     

A designed four-alpha-helix bundle that binds the volatile general anesthetic halothane with high affinity

The structural features of volatile anesthetic binding sites on proteins are being examined with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work has suggested that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein. The resulting bundle variant has an improved affinity (K(d) = 0.20 +/- 0.01 mM) for halothane binding, compared with the leucine-containing bundle (K(d) = 0.69 +/- 0.06 mM). Photoaffinity labeling with (14)C-halothane reveals preferential labeling of the W15 residue in both peptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-alpha-helix bundle proteins stabilized the native folded conformations. Molecular dynamics simulations of the bundles illustrate the existence of the hydrophobic core, containing both W15 residues. These results suggest that in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anesthetic action.     

Helical shifts generate two distinct conformers in the atomic resolution structure of the CheA phosphotransferase domain from Thermotoga maritima

Helical histidine phosphotransferase (HPt) domains play a central role in many aspects of bacterial signal transduction. The 0.98 A resolution crystallographic structure of the amino-terminal HPt domain (P1) from the chemotaxis kinase CheA of Thermotoga maritima reveals a remarkable degree of structural heterogeneity within a four-helix bundle. Two of the four helices have alternate main-chain conformations that differ by a 1.3-1.7A shift along the bundle axis. These dual conformers were only resolved with atomic resolution diffraction data and their inclusion significantly improved refinement statistics. Neither conformer optimizes packing within the helical core, consistent with their nearly equal refined occupancies. Altered hydrogen bonding within an inter-helical loop may facilitate transition between conformers. Two discrete structural states rather than a continuum of closely related conformations indicates an energetic barrier to conversion between conformers in the crystal at 100K, although many more states are expected in solution at physiological temperatures. Anisotropic atomic thermal B factors within the two conformers indicate modest overall atomic displacement that is largest perpendicular to the helical bundle and not along the direction of apparent motion. Despite the conformational heterogeneity of P1 in the crystal at low temperature, the protein displays high thermal stability in solution (T(m)=100 degrees C). Addition of a variable C-terminal region that corresponds to a mobile helix in other CheA structures significantly narrows the temperature width of the unfolding transition and may affect domain dynamics. Helices that compose the kinase recognition site and contain the phospho-accepting His45 do not have alternate conformations. In this region, atomic resolution provides detailed structural parameters for a conserved hydrogen-bonding network that tunes the reactivity of His45. A neighboring glutamate (E67), essential for phosphotransferase activity hydrogen bonds directly to His45 N(delta1). E67 generates a negative electrostatic surface surrounding the reactive His that is conserved by most CheA kinases, but absent in related phosphotransferase proteins. The P1 conformations that we observe are likely relevant to other helical or coiled-coil proteins and may be important for generating switches in signaling processes.     

Structure of Yeast Sulfhydryl Oxidase Erv1 Reveals Electron Transfer of the Disulfide Relay System in the Mitochondrial Intermembrane Space

The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX₉C or twin CX₃C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.     

Pressure stability of the alpha-helix structure in a de novo designed protein (alpha-l-alpha)(2) studied by FTIR spectroscopy

The pressure-induced structural changes of a de novo designed four-helix bundle protein, (alpha-l-alpha)(2), in aqueous solution have been investigated by FTIR spectroscopy. Changes in the amide I' band intensity show that pressure induces disruption of tertiary interactions and stabilizes the solvated alpha-helical form. This may suggest that the exposure of the hydrophobic core to the solvent by pressure is not a sufficient condition for pressure-induced unfolding of the alpha-helices of proteins.     

A model membrane protein for binding volatile anesthetics

Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4-3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism.     

Structural characterization and unfolding mechanism of human 4F2hc ectodomain

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, β1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% β-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (βα)(8) barrel; C, antiparallel β(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the β-barrel of domain A followed by additional hydrophobic interactions in domain C.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Human interleukin 4. The solution structure of a four-helix bundle protein.

Heteronuclear 13C and 15N three-dimensional nuclear magnetic resonance (n.m.r.) techniques have been used to determine the solution structure of human interleukin 4, a four-helix bundle protein. A dynamical simulated annealing protocol was used to calculate an ensemble of structures from an n.m.r. data set of 1735 distance restraints, 101 phi angle restraints and 27 pairs of hydrogen bond restraints. The protein structure has a left-handed up-up-down-down topology for the four helices with the two long overhand loops in the structure being connected by a short section of irregular antiparallel beta-sheet. Analysis of the side-chains in the protein shows a clustering of hydrophobic residues, particularly leucines, in the core of the bundle with the side-chains of charged residues being located on the protein surface. The solution structure has been compared with a recent structure prediction for human interleukin 4 and with crystal structures of other helix bundle proteins.     

Populating partially unfolded forms by hydrogen exchange-directed protein engineering

The native-state hydrogen exchange of a redesigned apocytochrome b(562) suggests that at least two partially unfolded forms (PUFs) exist for this four-helix bundle protein under native conditions. The more stable PUF has the N-terminal helix unfolded. To verify the conclusion further and obtain more detailed structural information about this PUF, five hydrophobic core residues in the N-terminal helix were mutated to Gly and Asp to destabilize the native state selectively and populate the PUF for structural studies. The secondary structure and the backbone dynamics of this mutant were characterized using multidimensional NMR. Consistent with the prediction, the N-terminal region of the mutant was found to be unfolded while other parts of the proteins remained folded. These results suggest that native-state hydrogen exchange-directed protein engineering can be a useful approach to populating partially unfolded forms for detailed structural studies.