The immunosuppressant drug cyclosporin A (CsA) inhibits T-cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high-affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes, pointing to fine structural differences in the calcineurin-binding region. To delineate the calcineurin-binding region of CyPB, we mutated several amino acids, located in two loops corresponding to CyPA regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type CyPB, the G77H, D155R, and D158R mutants had intact isomerase and CsA-binding activities, indicating that no major conformational changes had taken place. When complexed to CsA, they all displayed only reduced affinity for calcineurin and much decreased inhibition of calcineurin phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of CyPB/CsA with calcineurin, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in CyPA and might therefore account for the higher affinity of the CyPB/CsA complex for calcineurin.

A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for β-glycosidase from the hyperthermophilic bacterium Solfolobus sulfataricus (Sβgly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of Sβgly results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns (Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71–79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern–Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sβgly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sβlgy evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.

The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 Å resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded β/α-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.

The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu, Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen α1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.

We develop a protocol for estimating the free energy difference between different conformations of the same polypeptide chain. The conformational free energy evaluation combines the CHARMM force field with a continuum treatment of the solvent. In almost all cases studied, experimentally determined structures are predicted to be more stable than misfolded “decoys.” This is due in part to the fact that the Coulomb energy of the native protein is consistently lower than that of the decoys. The solvation free energy generally favors the decoys, although the total electrostatic free energy (sum of Coulomb and solvation terms) favors the native structure. The behavior of the solvation free energy is somewhat counterintuitive and, surprisingly, is not correlated with differences in the burial of polar area between native structures and decoys. Rather, the effect is due to a more favorable charge distribution in the native protein, which, as is discussed, will tend to decrease its interaction with the solvent. Our results thus suggest, in keeping with a number of recent studies, that electrostatic interactions may play an important role in determining the native topology of a folded protein. On this basis, a simplified scoring function is derived that combines a Coulomb term with a hydrophobic contact term. This function performs as well as the more complete free energy evaluation in distinguishing the native structure from misfolded decoys. Its computational efficiency suggests that it can be used in protein structure prediction applications, and that it provides a physically well-defined alternative to statistically derived scoring functions.

Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription. TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF). TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site. The solution structure of the N-terminal residues 2–59 of human TFIIB was determined by multidimensional NMR spectroscopy. The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and “rubredoxin” turns, three antiparallel β-strands, and disordered loops. The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (Pf TFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM). The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the β-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the β-sheet.

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains VH and VL. Both VH and VL adopt essentially native-like exchange protection within the dead time of the manual-mixing H/D exchange experiment (10 s) and in the case of VL, which contains two cis-prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than VL and VH. Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in VL, this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association.

A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1H NMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1Hα secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N-Cα-C=O) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06 ± 0.30 Å using residues V4–P35 and 0.51 ± 0.17 Å using residues A7–S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and “snorkeling” of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase.

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the α-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol−1 compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol−1 less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.

Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 Å resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein.

Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), and electron microscopy (EM) have been used simultaneously to follow the temperature-induced formation of amyloid fibrils by bovine insulin at acidic pH. The FTIR and CD data confirm that, before heating, insulin molecules in solution at pH 2.3 have a predominantly native-like α-helical structure. On heating to 70 °C, partial unfolding occurs and results initially in aggregates that are shown by CD and FT-IR spectra to retain a predominantly helical structure. Following this step, changes in the CD and FTIR spectra occur that are indicative of the extensive conversion of the molecular conformation from α-helical to β-sheet structure. At later stages, EM shows the development of fibrils with well-defined repetitive morphologies including structures with a periodic helical twist of ∼450 Å. The results indicate that formation of fibrils by insulin requires substantial unfolding of the native protein, and that the most highly ordered structures result from a slow evolution of the morphology of the initially formed fibrillar species.

The contributions of backbone NH group dynamics to the conformational heat capacity of the B1 domain of Streptococcal protein G have been estimated from the temperature dependence of 15N NMR-derived order parameters. Longitudinal (R1) and transverse (R2) relaxation rates, transverse cross-relaxation rates (ηxy), and steady state {1H}–15N nuclear Overhauser effects were measured at temperatures of 0, 10, 20, 30, 40, and 50 °C for 89–100% of the backbone secondary amide nitrogen nuclei in the B1 domain. The ratio R2/ηxy was used to identify nuclei for which conformational exchange makes a significant contribution to R2. Relaxation data were fit to the extended model-free dynamics formalism, incorporating an axially symmetric molecular rotational diffusion tensor. The temperature dependence of the order parameter (S2) was used to calculate the contribution of each NH group to conformational heat capacity (Cp) and a characteristic temperature (T*), representing the density of conformational energy states accessible to each NH group. The heat capacities of the secondary structure regions of the B1 domain are significantly higher than those of comparable regions of other proteins, whereas the heat capacities of less structured regions are similar to those in other proteins. The higher local heat capacities are estimated to contribute up to ∼0.8 kJ/mol K to the total heat capacity of the B1 domain, without which the denaturation temperature would be ∼9 °C lower (78 °C rather than 87 °C). Thus, variation of backbone conformational heat capacity of native proteins may be a novel mechanism that contributes to high temperature stabilization of proteins.

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N-terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, KD's are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of Gram-negative bacteria. These “Trojan Horse” antibiotics may prove useful as an efficient means to combat multi-drug–resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.

An analysis of the folding of the 94 residue tenth fibronectin type III (fnIII) domain of human fibronectin (FNfn10) is presented. Use of guanidine isothiocyanate as a denaturant allows us to obtain equilibrium and kinetic data across a broad range of denaturant concentrations that are unavailable in guanidine hydrochloride. Equilibrium unfolding experiments show that FNfn10 is significantly more stable than has been reported previously. Comparison of equilibrium and kinetic parameters reveals the presence of an intermediate that accumulates at low denaturant concentrations. This is the first demonstration of three-state folding kinetics for a fnIII domain. We have previously shown that a homologous domain from human tenascin (TNfn3) folds by a two-state mechanism, but this does not necessarily indicate that the two proteins fold by different folding pathways.

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended β-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.

The nitrogenase enzyme of Klebsiella pneumoniae consists of two separable proteins, each with multiple subunits and one or more oxygen sensitive metallocenters. The wild-type nitrogenase proteins are stable to electrophoresis in high concentrations of urea under anaerobic conditions. Addition of Mg+2 and ADP greatly increases the stability of the smaller Fe protein (from <4 to >6 M for full unfolding), an effect directly analogous to stabilization in p21ras induced by Mg+2 and GDP. Stabilization by Mg+2 is slight for the holo MoFe protein (from ∼1.5 to ∼2.4 M) but more dramatic for the apo protein form of the MoFe protein accumulated by certain Fe protein (nifH gene) mutants. The potent product inhibitor of nitrogenase function, MgADP, increases stability of the MoFe protein more than Mg+2 alone, to ∼3.6 M, showing that nucleotides interact with the MoFe protein. Mutations of the nifM gene result in slower accumulation of less stable Fe protein, indicating that NifM is involved in correct folding of the Fe protein. Mutationally altered proteins are often difficult to purify for study because of their inherent instability, low expression level, or oxygen lability. Crude extracts of 11 different mutants of Fe protein (nifH gene) were examined by transverse urea gradient gels to rapidly screen for stabilizing interactions in the presence or absence of substrate or inhibitor analogs. Amino acid alterations D44N and R188C, at the interface of the dimer, in the vicinity of the nucleotide binding site(s), have significantly lower stability than the wild-type enzyme in the absence of Mg+2 but comparable stability in its presence, showing the importance of Mg+2 in the subunit interactions. Mutations N163S and E266K, in which residues normally involved in hydrogen bonding far from the active site were altered, are more labile than the wild-type even with Mg+2 added. Seven other mutants, though nonfunctional, did not appear altered in stability compared to the wild-type.

One of the proposed roles of the GroEL-GroES cavity is to provide an “infinite dilution” folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these “nonpermissive” folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these “low Mg2+” conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

Integral membrane proteins carry out some of the most important functions of living cells, yet relatively few details are known about their structures. This is due, in large part, to the difficulties associated with preparing membrane protein crystals suitable for X-ray diffraction analysis. Mechanistic studies of membrane protein crystallization may provide insights that will aid in determining future membrane protein structures. Accordingly, the solution behavior of the bacterial outer membrane protein OmpF porin was studied by static light scattering under conditions favorable for crystal growth. The second osmotic virial coefficient (B22) was found to be a predictor of the crystallization behavior of porin, as has previously been found for soluble proteins. Both tetragonal and trigonal porin crystals were found to form only within a narrow window of B22 values located at approximately −0.5 to −2 × 10−4 mol mL g−2, which is similar to the “crystallization slot” observed for soluble proteins. The B22 behavior of protein-free detergent micelles proved very similar to that of porin-detergent complexes, suggesting that the detergent's contribution dominates the behavior of protein-detergent complexes under crystallizing conditions. This observation implies that, for any given detergent, it may be possible to construct membrane protein crystallization screens of general utility by manipulating the solution properties so as to drive detergent B22 values into the crystallization slot. Such screens would limit the screening effort to the detergent systems most likely to yield crystals, thereby minimizing protein requirements and improving productivity.

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as α-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like α-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of HL from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate ∼0.1 s−1, which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.