Backbone dynamics of the basic/helix-loop-helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA. Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states. These secondary structures are compared to the co-crystal complex of Pho4 bound to a cognate DNA sequence (Shimizu T, Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Hakoshima T, 1997, EMBO J 15: 4689–4697). The dynamic information provides insight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex. Relative to the unbound form, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region. The nonspecific and cognate complexes, however, have nearly identical secondary structure and backbone dynamics. We also present evidence for conformational flexibility at a highly conserved glutamate basic region residue. These results are discussed in relation to the mechanism of sequence specific recognition and binding.

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of chemical exchange cross peaks in 750 MHz 15N–1H–15N HSQC–NOESY–HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange. The rate of exchange from the major to the minor form was determined to be 1.1 ± 0.2 s−1 at 298 K. Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 ± 0.11 s−1 at 288 K, and that the bound Im9 is fixed in one conformation. The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 Å radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange. The possible functional significance of this feature of the DNase is discussed.

The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3′-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a “head-to-tail” arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (± thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (± thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 °C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 °C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.

Parvalbumins constitute a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7:2397–2408), we presented the sequence of the major parvalbumin isoform from the silver hake (Merluccius bilinearis) and presented spectroscopic and structural information on the excised “EF-hand” portion of the protein. In this study, the X-ray crystal structure of the silver hake major parvalbumin has been determined to high resolution, in the frozen state, using the molecular replacement method with the carp parvalbumin structure as a starting model. The crystals are orthorhombic, space group C2221, with a = 75.7 Å, b = 80.7 Å, and c = 42.1 Å. Data were collected from a single crystal grown in 15% glycerol, which served as a cryoprotectant for flash freezing at −188 °C. The structure refined to a conventional R-value of 21% (free R 25%) for observed reflections in the range 8 to 1.65 Å [I > 2σ(I)]. The refined model includes an acetylated amino terminus, 108 residues (characteristic of a β parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simulations focused primarily on the dynamics of the ligands coordinating the Ca2+ ions in the CD and EF sites. MD simulations were performed on both the fully Ca2+ loaded protein and on a Ca2+ deficient variant, with Ca2+ only in the CD site. There was substantial agreement between the MD and X-ray results in addressing the issue of mobility of key residues in the calcium-binding sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site.

Understanding protein folding requires the determination of the configurational space accessible to the protein at different stages in folding. Here, computer simulation analysis of small angle neutron scattering results is used to probe the change in the distribution of configurations on strong denaturation of a globular protein, phosphoglycerate kinase, in 4 M guanidine hydrochloride solution. To do this atomic-detail ensembles of the unfolded protein chain are modeled and their scattering profiles compared with the experiment. The local conformational statistics are found to strongly influence the experimental intensity at scattering vectors between 0.05 and 0.3 Å−1. Denaturation leads to a reduction in the protein atom-pair distance distribution function over the ∼3–15 Å region that is associated with a quantifiable shift in the backbone torsional angle (φ,ψ) distribution toward the β region of the Ramachandran plot.

The formation of the N-terminal β-hairpin of ubiquitin is thought to be an early event in the folding of this small protein. Previously, we have shown that a peptide corresponding to residues 1–17 of ubiquitin folds autonomously and is likely to have a native-like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U(1–17)T9D, the native conformation is stabilized with respect to the wild-type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U(1–17)T9D peptide does indeed form a hairpin with a native-like register and a type I turn with a G1 β-bulge, as in the full-length protein. The reason for the greater stability of the U(1–17)T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys11, which may indicate that a charge–charge interaction between these residues is responsible.

Biotin and lipoic acid moieties are the covalently attached coenzyme cofactors of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of these moieties to the biotinyl- and lipoyl-dependent enzymes is post-translationally catalyzed by specific biotinylating and lipoylating protein enzymes. In Escherichia coli, two different enzymes, LplA and LipB, catalyze independent pathways for the lipoylation of the relevant enzymes, whereas only one enzyme, the BirA protein, is responsible for all the biotinylation. Counterparts of the E. coli BirA, LplA, and LipB enzymes have been previously identified in many organisms, but homology among the three families has never been reported. Computational analysis based on PSI-BLAST profiles and secondary structure predictions indicates, however, that lipoylating and biotinylating enzymes are evolutionarily related protein families containing a homologous catalytic module. Sequence conservation among the three families is very poor, but a single lysine residue is strictly conserved in all of them, which, according to the available X-ray crystal structure of the E. coli BirA protein, is expected to contribute to the binding of lipoic acid in the LplA and LipB enzymes.

The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.

Previous conformational analysis of 10-residue linear peptides enabled us to identify some cross-strand side-chain interactions that stabilize β-hairpin conformations. The stabilizing influence of these interactions appeared to be greatly reduced when the interaction was located at the N- and C-termini of these 10-residue peptides. To investigate the effect of the position relative to the turn of favorable interactions on β-hairpin formation, we have designed two 15-residue β-hairpin forming peptides with the same residue composition and differing only in the location of two residues within the strand region. The conformational properties of these two peptides in aqueous solution were studied by 1H and 13C NMR. Differences in the conformational behavior of the two designed 15-residue peptides suggest that the influence of stabilizing factors for β-hairpin formation, in particular, cross-strand side-chain interactions, depends on their proximity to the turn. Residues adjacent to the turn are most efficient in that concern. This result agrees with the proposal that the turn region acts as the driving force in β-hairpin folding.

Bovine β-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4–2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4–5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low ΔG°20 values of ∼2.7 (N-domain) and ∼1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20–25 °C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] ≥0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities.

For the intact protein (±Ca2+), the bimodal denaturation profiles can be analyzed to give two ΔG°20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.

The WW domain is an approximately 38 residue peptide-binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one-third of its three-stranded antiparallel β-sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high-affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild-type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3–84 were 0.76 ± 0.14 Å for the backbone atoms and 1.36 ± 0.15 Å for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 Å, respectively, for residues 4–83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H–1H–13C NOESY-HSQC spectra with and without 13C decoupling. The NMR-defined interface agrees well with that determined in a recent X-ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.

The heat of binding the serine protease, porcine pancreatic elastase, by the inhibitor, turkey ovomucoid third domain, is dependent on the presence of inorganic phosphate. This dependence is saturable and can be accurately modeled as the phosphate binding to a single site on the protease–inhibitor complex; thus, the elastase–ovomucoid system provides a unique opportunity to study phosphate–protein interactions. We have used isothermal titration calorimetry to investigate this binding, thereby providing one of the few complete thermodynamic characterizations of phosphate interacting with proteins. The binding is characterized by a small favorable ΔG°, a large unfavorable ΔH°, and a positive ΔCp, thermodynamics consistent with the release of water being linked to phosphate binding. These measurements provide insight into the binding of phosphotyrosine containing peptides to SH2 domains by suggesting the energetic consequences of binding phosphate free from other interactions.

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (α, ε, and θ). The θ subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the θ subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, θ was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3JHNα coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10–Asp19, Glu38–Glu43, and His47–Glu54), one short extended segment (Pro34–Ala37), and a long loop (Ala20–Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of θ is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the ε subunit have identified a loop region (Ala20–Glu29) in θ as the site of association with ε.

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 Å were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 Å from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds.

This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process.

This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26–Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control.

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three β-branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 Å resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 Å resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The β-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type γ-branched P1 residue for OMTKY3, Leu18I. χ1 angles of approximately 40° are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60°, −60°, or 180° are more commonly observed but 40° is not unfavorable for the β-branched side chains. Thr18I Oγ1 also forms a hydrogen bond with Ser195 Oγ in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.

We have identified a similarity between the apical domain of the human transferrin receptor and several other protein families. This domain is found associated with two different families of peptidases. Therefore, we term it the PA domain for protease-associated domain. The PA domain is found inserted within a loop of the peptidase domain of family M8/M33 zinc peptidases. The PA domain is also found in a vacuolar sorting receptor and a ring finger protein of unknown function that may be a cell surface receptor. The PA domain may mediate substrate determination of peptidases or form protein–protein interactions.