NMR characterization of residual structure in the denatured state of protein L

Triple-resonance NMR experiments were used to assign the (13)C(alpha), (13)C(beta), (15)N and NH resonances for all the residues in the denatured state of a destabilized protein L variant in 2 M guanidine. The chemical shifts of most resonances were very close to their random coil values. Significant deviations were observed for G22, L38 and K39; increasing the denaturant concentration shifted the chemical shifts of these residues towards theory random coil values. Medium-range nuclear Overhauser enhancements were detected in segments corresponding to the turn between the first two strands, the end of the second strand through the turn between the second strand and the helix, and the turn between the helix and the third strand in 3D H(1), N(15)-HSQC-NOESY-HSQC experiments on perdeuterated samples. Longer-range interactions were probed by measuring the paramagnetic relaxation enhancement produced by nitroxide spin labels introduced via cysteine residues at five sites around the molecule. Damped oscillations in the magnitude of the paramagnetic relaxation enhancement as a function of distance along the sequence suggested native-like chain reversals in the same three turn regions. The more extensive interactions within the region corresponding to the first beta-turn than in the region corresponding to the second beta-turn suggests that the asymmetry in the folding reaction evident in previous studies of the protein L folding transition state is already established in the denatured state.     

NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.     

Characterisation of the conformational properties of urea-unfolded Im7: implications for the early stages of protein folding

The colicin immunity protein Im7 folds from its unfolded state in 6 M urea to its native four-helix structure through an on-pathway intermediate that lacks one of the helices of the native structure (helix III). In order to further characterize the folding mechanism of Im7, we have studied the conformational properties of the protein unfolded in 6 M urea in detail using heteronuclear NMR. Triple-resonance experiments with 13C/15N-labelled Im7 in 6 M urea provided almost complete resonance assignments for the backbone nuclei, and measurement of backbone 15N relaxation parameters allowed dynamic ordering of the unfolded polypeptide chain to be investigated. Reduced spectral density mapping and fitting backbone R2 relaxation rates to a polymer dynamics model identified four clusters of interacting residues, each predicted by the average area buried upon folding for each residue. Chemical shift analyses and measurement of NOEs detected with a long mixing-time 1H-1H-15N NOESY-HSQC spectrum confirmed the formation of four clusters. Each cluster of interacting side-chains in urea-unfolded Im7 occurs in a region of the protein that forms a helix in the protein, with the largest clusters being associated with the three long helices that are formed in the on-pathway folding intermediate, whilst the smallest cluster forms a helix only in the native state. NMR studies of a Phe15Ala Im7 variant and a protein in which residues 51-56 are replaced by three glycine residues (H3G3 Im7*), indicated that the clusters do not interact with each other, possibly because they are solvated by urea, as indicated by analysis of NOEs between the protein and the solvent. Based on these data, we suggest that dilution of the chaotrope to initiate refolding will result in collapse of the clusters, leading to the formation of persistent helical structure and the generation of the three-helix folding intermediate.     

Multistep denaturation of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi, is a dumbbell-shaped protein in which two globular domains are connected by a three-stranded beta-sheet segment that is solvent-exposed on both faces. Previous studies showed that the whole protein, including the single-layer beta-sheet, is highly rigid. To elucidate the folding mechanism and the role of the central beta-sheet in the formation of the rigid molecule, we investigated the equilibrium thermal denaturation reaction of OspA. We applied differential scanning calorimetry, heteronuclear NMR spectroscopy, and solution small-angle X-ray scattering (SAXS) to characterize the reaction in detail. All three techniques revealed that OspA denatures in two separable cooperative transitions. NMR measurements on OspA specifically 15N-labeled at Lys residues identified the locations of the two folding units and revealed that the C-terminal segment is less stable than the remaining N-terminal segment. The boundary between the two folding units is located within the central beta-sheet. The interconversion among the three folding states (fully folded, C-terminus unfolded, and fully denatured) is slow relative to chemical shift differences (<24 Hz), indicating that there are significant kinetic barriers in the denaturation reactions. SAXS measurements determined the radius of gyration of the native protein to be 25.0 +/- 0.3 A, which increases to 34.4 +/- 1.0 A in the first transition, and then to 56.1 +/- 1.6 A in the second transition. Thus, the intermediate state, in which the C-terminal folding unit is already denatured, is still compact. These results provide a basis for elucidating the folding mechanism of OspA.     

Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin.

Bovine beta-lactoglobulin is denatured by increased temperature (heat denaturation) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured state of beta-lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD and SAXS indicated that the cold-denatured state, in comparison with the highly denatured state induced by urea, is rather compact, retaining some secondary structure, but no tertiary structure. The location of the residual structures in the cold-denatured state and their stability were characterized by 1H/2H exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting beta-strands G and H had markedly high protection factors, suggesting the presence of a native-like beta-hairpin stabilized by the disulfide bond. Since this beta-hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount importance for the folding and stability of beta-lactoglobulin. On the other hand, the non-native alpha-helix suggested for the folding intermediate was not detected in the cold-denatured state. The 1H/2H exchange experiments showed that the protection factors of a mixture of the native and cold-denatured states is strongly biased by that of the labile cold-denatured state, consistent with a two-process model of the exchange.     

NMR structure of an F-actin-binding "headpiece" motif from villin

A growing family of F-actin-bundling proteins harbors a modular F-actin-binding headpiece domain at the C terminus. Headpiece provides one of the two F-actin-binding sites essential for filament bundling. Here, we report the first structure of a functional headpiece domain. The NMR structure of chicken villin headpiece (HP67) reveals two subdomains that share a tightly packed hydrophobic core. The N-terminal subdomain contains bends, turns, and a four-residue alpha-helix as well as a buried histidine residue that imparts a pH-dependent folding. The C-terminal subdomain is composed of three alpha-helices and its folding is pH-independent. Two residues previously implicated in F-actin-binding form a buried salt-bridge between the N and C-terminal subdomains. The rest of the identified actin-binding residues are solvent-exposed and map onto a unique F-actin-binding surface.     

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Plastocyanin is a predominantly beta-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,Halpha) coupling constants, (15)N-edited proton-proton nuclear Overhauser effects (NOEs), and (15)N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to beta-strands in the folded protein exhibit small upfield shifts of the (13)C(alpha) and (13)CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i + 1) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the (13)C(alpha), (13)CO, and (1)H(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,Halpha) coupling constants. The (15)N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form beta-strands in the native protein spontaneously populate the beta-region of (phi,psi) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.     

Effect of subdomain interactions on methyl group dynamics in the hydrophobic core of villin headpiece protein

Thermostable villin headpiece protein (HP67) consists of the N‐terminal subdomain (residues 10–41) and the autonomously folding C‐terminal subdomain (residues 42–76) which pack against each other to form a structure with a unified hydrophobic core. The X‐ray structures of the isolated C‐terminal subdomain (HP36) and its counterpart in HP67 are very similar for the hydrophobic core residues. However, fine rearrangements of the free energy landscape are expected to occur because of the interactions between the two subdomains. We detect and characterize these changes by comparing the µs‐ms time scale dynamics of the methyl‐bearing side chains in isolated HP36 and in HP67. Specifically, we probe three hydrophobic side chains at the interface of the two subdomains (L42, V50, and L75) as well as at two residues far from the interface (L61 and L69). Solid‐state deuteron NMR techniques are combined with computational modeling for the detailed characterization of motional modes in terms of their kinetic and thermodynamic parameters. The effect of interdomain interactions on side chain dynamics is seen for all residues but L75. Thus, changes in dynamics because of subdomain interactions are not confined to the site of perturbation. One of the main results is a two‐ to threefold increase in the value of the activation energies for the rotameric mode of motions in HP67 compared with HP36. Detailed analysis of configurational entropies and heat capacities complement the kinetic view of the degree of the disorder in the folded state.     

Electrostatic screening and backbone preferences of amino acid residues in urea-denatured ubiquitin

Local structures in denatured proteins may be important in guiding a polypeptide chain during the folding and misfolding processes. Existence of local structures in chemically denatured proteins is a highly controversial issue. NMR parameters [coupling constants (3) J(H(alpha),H(N)) and chemical shifts] of chemically denatured proteins in general deviate little from their values in small peptides. These peptides were presumed to be completely unstructured; therefore, it was considered that chemically denatured proteins are random coils. But recent experimental studies show that small peptides adopt relatively stable structures in aqueous solutions. Small deviations of the NMR parameters from their values in small peptides may thus actually indicate the existence of local structures in chemically denatured proteins. Using NMR data and theoretical predictions we show here that fluctuating beta-strands exist in urea-denatured ubiquitin (8 M urea at pH 2). Residues in such beta-strands populate more frequently the left side of the broad beta region of -psi space. Urea-denatured ubiquitin contains no detectable beta-sheet secondary structures; nevertheless, the fluctuating beta-strands in urea-denatured ubiquitin coincide to the beta-strands in the native state. Formation of beta-strands is in accord with the electrostatic screening model of unfolded proteins. The free energy of a residue in an unfolded protein is in this model determined by the local backbone electrostatics and its screening by backbone solvation. These energy terms introduce strong electrostatic coupling between neighboring residues, which causes cooperative formation of beta-strands in denatured proteins. We propose that fluctuating beta-strands in denatured proteins may serve as initiation sites to form fibrils.     

Native and nonnative conformational preferences in the urea-unfolded state of barstar

The refolding of barstar from its urea-unfolded state has been studied extensively using various spectroscopic probes and real-time NMR, which provide global and residue-specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25 degrees C. Complete backbone resonance assignments of the urea-unfolded protein were obtained using the recently developed three-dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13C(alpha) and 13CO secondary shifts show positive deviations in the urea-unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to beta-strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the beta-strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.     

NMR-detected order in core residues of denatured bovine pancreatic trypsin inhibitor

The NMR characteristics of [14-38]Abu, a synthetic variant of BPTI that is partially folded in aqueous buffer near neutral pH, support a model of early folding events which begin with stabilization of the nativelike, slow exchange core [Barbar, E., Hare, M., Daragan, V., Barany, G., and Woodward, C. (1998) Biochemistry 37, 7822-7833 (1)]. In partially folded [14-38]Abu, urea denaturation profiles for representative amide protons show that global unfolding is non-two-state and that core residues require a higher concentration of urea to unfold. Dynamic properties of pH-denatured [14-38]Abu and fully reduced and unfolded BPTI analogue were determined from heteronuclear NMR relaxation measurements at similar solution conditions. Differences at various sites in the polypeptide chain were evaluated from spectral density functions determined from T1, T2, and steady-state heteronuclear NOE data. Although denatured [14-38]Abu contains no persistent secondary structure, its most ordered residues are those that, in native BPTI, fold into the slow exchange core. The fully reduced analogue is significantly more mobile and shows less heterogeneous dynamics, but at 1 degree C, restricted motion is observed for residues in the central segments of the polypeptide chain. These observations indicate that there is a developing core or cores even in highly unfolded species. Apparently the effect of 14-38 disulfide on unfolded     

Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy

Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop. To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy. Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure. This Ca(2+)-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and H(alpha) chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif.     

Side-chain conformations in an unfolded protein: chi1 distributions in denatured hen lysozyme determined by heteronuclear 13C, 15N NMR spectroscopy.

Using a 13C and 15N-labelled sample, multi-dimensional heteronuclear NMR techniques have been carried out to characterise hen lysozyme denatured in 8 M urea at pH 2.0. The measurement of 3J(C',Cgamma) and 3J(N,Cgamma) coupling constants has enabled side-chain chi1 torsion angle populations to be probed in the denatured polypeptide chain. Analysis of the coupling constant data has allowed the relative populations of the three staggered rotamers about chi1 to be defined for 51 residues. The amino acids can broadly be divided into five classes that show differing side-chain conformational preferences in the denatured state. These range from a strong preference for the -60 degrees chi1 rotamer for methionine and leucine (74-79 % population) to a favouring of the +60 degrees chi1 rotamer for threonine (67 % population). The differences in behaviour reflect the steric and electrostatic characteristics of the side-chains concerned. A close agreement is seen between the chi1 populations calculated from the experimental coupling constant data and predictions from the statistical model for a random coil that uses the chi1 torsion angle distributions in a data base of native protein structures. Short-range interactions therefore dominate in determining the local conformational properties of side-chains in a denatured protein. Deviations are, however, observed for many of the aromatic residues involved in hydrophobic clusters within the denatured protein. For these residues the effects of additional non-local interactions in the clusters presumably play a major role in determining the chi1 preferences.     

Detection of initiation sites in protein folding of the four helix bundle ACBP by chemical shift analysis

A simple alternative method for obtaining "random coil" chemical shifts by intrinsic referencing using the protein's own peptide sequence is presented. These intrinsic random coil backbone shifts were then used to calculate secondary chemical shifts, that provide important information on the residual secondary structure elements in the acid-denatured state of an acyl-coenzyme A binding protein. This method reveals a clear correlation between the carbon secondary chemical shifts and the amide secondary chemical shifts 3-5 residues away in the primary sequence. These findings strongly suggest transient formation of short helix-like segments, and identify unique sequence segments important for protein folding.     

Peptide models provide evidence for significant structure in the denatured state of a rapidly folding protein: the villin headpiece subdomain

The villin headpiece subdomain is a cooperatively folded 36-residue, three-alpha-helix protein. The domain is one of the smallest naturally occurring sequences which has been shown to fold. Recent experimental studies have shown that it folds on the 10-micros time scale. Its small size, simple topology, and very rapid folding have made it an attractive target for computational studies of protein folding. We present temperature-dependent NMR studies that provide evidence for significant structure in the denatured state of the headpiece subdomain. A set of peptide fragments derived from the headpiece were also characterized in order to determine if there is a significant tendency to form a locally stabilized structure in the denatured state. Peptides corresponding to each of the three isolated helices and to the connection between the first and second helices were largely unstructured. A longer peptide fragment which contains the first and second helices shows considerable structure, as judged by NMR and CD. Concentration-dependent CD measurements and analytical ultracentrifugation experiments indicate that the structure is not due to self-association. NMR studies indicate that the structure is stabilized by tertiary interactions involving phenylalanines and Val 50. A peptide in which two of the three phenylalanines are changed to leucine is considerably less structured, confirming the importance of the phenylalanines. This work indicates that there is significant structure in the denatured state of this rapidly folding protein.     

Structure and dynamics of an acid-denatured protein G mutant

NMR studies of protein denatured states provide insights into potential initiation sites for folding that may be too transient to be observed kinetically. We have characterized the structure and dynamics of the acid-denatured state of protein G by using a F30H mutant of G(B1) which is on the margin of stability. At 5 degrees C, F30H-G(B1) is greater than 95% folded at pH 7.0 and is greater than 95% unfolded at pH 4.0. This range of stability is useful because the denatured state can be examined under relatively mild conditions which are optimal for folding G(B1). We have assigned almost all backbone (15)N, H(N), and H(alpha) resonances in the acid-denatured state. Chemical shift, coupling constant, and NOE data indicate that the denatured state has considerably more residual structure when studied under these mild conditions than in the presence of chemical denaturants. The acid-denatured state populates nativelike conformations with both alpha-helical and beta-hairpin characteristics. To our knowledge, this is the first example of a denatured state with NOE and coupling constant evidence for beta-hairpin character. A number of non-native turn structures are also detected, particularly in the region corresponding to the beta1-beta2 hairpin of the folded state. Steady-state ?(1)H-(15)N? NOE results demonstrate restricted backbone flexibility in more structured regions of the denatured protein. Overall, our studies suggest that regions of the helix, the beta3-beta4 hairpin, and the beta1-beta2 turn may serve as potential initiation sites for folding of G(B). Furthermore, residual structure in acid-denatured F30H-G(B1) is more extensive than in peptide fragments corresponding to the beta1-beta2, alpha-helix, and beta3-beta4 regions, suggesting additional medium-to-long-range interactions in the full-length polypeptide chain.     

Sensitivity of NMR residual dipolar couplings to perturbations in folded and denatured staphylococcal nuclease

The invariance of NMR residual dipolar couplings (RDCs) in denatured forms of staphylococcal nuclease to changes in denaturant concentration or amino acid sequence has previously been attributed to the robustness of long-range structure in the denatured state. Here we compare RDCs of the wild-type nuclease with those of a fragment that retains a folded OB-fold subdomain structure despite missing the last 47 of 149 residues. The RDCs of the intact protein and of the truncation fragment are substantially different under conditions that favor folded structure. By contrast, there is a strong correlation between the RDCs of the full-length protein and the fragment under denaturing conditions (6 M urea). The RDCs of the folded and unfolded forms of the proteins are uncorrelated. Our results suggest that RDCs are more sensitive to structural changes in folded than unfolded proteins. We propose that the greater susceptibility of RDCs in folded states is a consequence of the close packing of the polypeptide chain under native conditions. By contrast, the invariance of RDCs in denatured states is more consistent with a disruption of cooperative structure than with the retention of a unique long-range folding topology.     

Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding

Villin-type headpiece domains are ～70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.     

Nonrandom structure in the urea-unfolded Escherichia coli outer membrane protein X (OmpX)

On the basis of sequence-specific resonance assignments for the complete polypeptide backbone and most of the amino acid side chains by heteronuclear nuclear magnetic resonance (NMR) spectroscopy, the urea-unfolded form of the outer membrane protein X (OmpX) from Escherichia coli has been structurally characterized. (1)H-(1)H nuclear Overhauser effects (NOEs), dispersion of the chemical shifts, amide proton chemical shift temperature coefficients, amide proton exchange rates, and (15)N[(1)H]-NOEs show that OmpX in 8 M urea at pH 6.5 is globally unfolded, but adopts local nonrandom conformations in the polypeptide segments of residues 73-82 and 137-145. For these two regions, numerous medium-range and longer-range NOEs were observed, which were used as the input for structure calculations of these polypeptide segments with the program DYANA. The segment 73-82 forms a quite regular helical structure, with only loosely constrained amino acid side chains. In the segment 137-145, the tryptophan residue 140 forms the core of a small hydrophobic cluster. Both nonrandom structures are present with an abundance of about 25% of the protein molecules. The sequence-specific NMR assignment and the physicochemical characterization of urea-denatured OmpX presented in this paper are currently used as a platform for investigations of the folding mechanism of this integral membrane protein.