The tumor suppressor p53 is conformationally unstable at physiological temperature. Even the activated p53Δ30 variant, which lacks the self-inhibiting carboxy terminal domain, has a half-life of only 8 min at 37 °C in vitro. We have developed a genetic approach to identify p53 variants that stabilize the active conformation. The human p53Δ30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein. Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site. The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening. The DNA binding activity of N239Y/N268D/E336V p53Δ30 variant has a half-life of 100 min at 37 °C, 12 times longer than that of the parental protein. The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild-type protein.

The effect of pressure on amide 15N chemical shifts was studied in uniformly 15N-labeled basic pancreatic trypsin inhibitor (BPTI) in 90%1H2O/10%2H2O, pH 4.6, by 1H-15N heteronuclear correlation spectroscopy between 1 and 2,000 bar. Most 15N signals were low field shifted linearly and reversibly with pressure (0.468 ± 0.285 ppm/2 kbar), indicating that the entire polypeptide backbone structure is sensitive to pressure. A significant variation of shifts among different amide groups (0–1.5 ppm/2 kbar) indicates a heterogeneous response throughout within the three-dimensional structure of the protein. A tendency toward low field shifts is correlated with a decrease in hydrogen bond distance on the order of 0.03 Å/2 kbar for the bond between the amide nitrogen atom and the oxygen atom of either carbonyl or water. The variation of 15N shifts is considered to reflect site-specific changes in φ, ψ angles. For β-sheet residues, a decrease in ψ angles by 1–2°/2 kbar is estimated. On average, shifts are larger for helical and loop regions (0.553 ± 0.343 and 0.519 ± 0.261 ppm/2 kbar, respectively) than for β-sheet (0.295 ± 0.195 ppm/2 kbar), suggesting that the pressure-induced structural changes (local compressibilities) are larger in helical and loop regions than in β-sheet. Because compressibility is correlated with volume fluctuation, the result is taken to indicate that the volume fluctuation is larger in helical and loop regions than in β-sheet. An important aspect of the volume fluctuation inferred from pressure shifts is that they include motions in slower time ranges (less than milliseconds) in which many biological processes may take place.

This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy.

The analogue is [d-AlaB26]des-(B27–B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of d-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length.

The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the “active” conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [d-AlaB26]des-(B27–B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.

We report the 0.75 Å crystal structure of a racemic mixture of the 12-residue designed peptide “Alpha-1” (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1. The unit cell contains two l-alpha-helices and two d-alpha-helices. The columnar-sheet bilayer motif seen in l-Alpha-1 is maintained in the d,l-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the l-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild-type and seven site-specific charge-reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein–protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein–protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.

Based on results from both equilibrium and kinetic hydrogen exchange studies of Escherichia coli ribonuclease HI (RNase H), a fragment of RNase H (eABCD) was designed. The sequence of eABCD contains less than half of the protein's primary sequence and includes the regions that were shown to be the most protected from hydrogen exchange in all previous studies of RNase H. This core fragment of RNase H encodes a well-ordered protein with native-like properties. When isolated from the full-length monomeric protein, the eABCD fragment forms a stable dimer. However, we show indirectly that the monomeric form of eABCD is folded and has an overall secondary structure similar to the dimeric form.

Fructose 1,6-bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6-bisphosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the ε-amino group of Lys229. The existing apo-enzyme structure was refined using the crystallographic free-R-factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6-bisphosphate), and the crystal structure of this complex has been determined to 2.8 Å. The apo structure differs from the previous Brookhaven-deposited structure (1ald) in the flexible C-terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen-bonded Michaelis complex before the formation of the covalent complex.

The pokeweed antiviral protein (PAP) belongs to a family of ribosome-inactivating proteins (RIP), which depurinate ribosomal RNA through their site-specific N-glycosidase activity. We report low temperature, three-dimensional structures of PAP co-crystallized with adenyl-guanosine (ApG) and adenyl-cytosine-cytosine (ApCpC). Crystal structures of 2.0–2.1 Å resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome-inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N-glycosidase activity of PAP toward adenine-containing single stranded RNA. We also report the analysis of a 2.1 Å crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 180° rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135.

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and elethylene glycol as an uncompetitve inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

The refolding of four disulfide lysozyme (at pH 5.2, 20 °C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the α- and β-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 °C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths β-domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near-native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.

Excitable cells and tissues like muscle or brain show a highly fluctuating consumption of ATP, which is efficiently regenerated from a large pool of phosphocreatine by the enzyme creatine kinase (CK). The enzyme exists in tissue—as well as compartment-specific isoforms. Numerous pathologies are related to the CK system: CK is found to be overexpressed in a wide range of solid tumors, whereas functional impairment of CK leads to a deterioration in energy metabolism, which is phenotypic for many neurodegenerative and age-related diseases. The crystal structure of chicken cytosolic brain-type creatine kinase (BB-CK) has been solved to 1.41 Å resolution by molecular replacement. It represents the most accurately determined structure in the family of guanidino kinases. Except for the N-terminal region (2–12), the structures of both monomers in the biological dimer are very similar and closely resemble those of the other known structures in the family. Specific Ca2+-mediated interactions, found between two dimers in the asymmetric unit, result in structurally independent heterodimers differing in their N-terminal conformation and secondary structure.

The high-resolution structure of BB-CK presented in this work will assist in designing new experiments to reveal the molecular basis of the multiple isoform-specific properties of CK, especially regarding different subcellular locations and functional interactions with other proteins. The rather similar fold shared by all known guanidino kinase structures suggests a model for the transition state complex of BB-CK analogous to the one of arginine kinase (AK). Accordingly, we have modeled a putative conformation of CK in the transition state that requires a rigid body movement of the entire N-terminal domain by rms 4 Å from the structure without substrates.

Immunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well-known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv-binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3–4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5 ± 0.5 × 105 M−1. A synthetic single immunoglobulin binding domain, Z-domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E-domain with hu4D5 Fab are n = 1.0 ± 0.1, Ka = 2.0 ± 0.3 × 105 M−1, and ΔH = −7.1 ± 0.4 kcal mol−1. Similar binding thermodynamics are obtained for titration of the isolated VH domain with E-domain indicating that the E-domain binding site on Fab resides within VH. E-domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, Ka > 1.0 × 107 M−1, and ΔH = −24.6 ± 0.6 kcal mol−1. Fc does not compete with Fab for binding to E-domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix-2 and helix-3 of E-domain, distinct from the Fc-binding site observed in the crystal structure of the B-domain/Fc complex. The Fv-binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the VH domain implicated previously in protein A binding.

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant κ4 light-chain variable domains (VLs) derived from amyloidogenic light chains with a VL from a benign light chain. The amyloidogenic VLs were significantly less stable than the benign VL. Furthermore, only the amyloidogenic VLs formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic VLs on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign VL could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign VL that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the VL β-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.

The sterile alpha motif (SAM) is a protein interaction domain of around 70 amino acids present predominantly in the N- and C-termini of more than 60 diverse proteins that participate in signal transduction and transcriptional repression. SAM domains have been shown to homo- and hetero-oligomerize and to mediate specific protein–protein interactions. A highly conserved subclass of SAM domains is present at the intracellular C-terminus of more than 40 Eph receptor tyrosine kinases that are involved in the control of axonal pathfinding upon ephrin-induced oligomerization and activation in the event of cell–cell contacts. These SAM domains appear to participate in downstream signaling events via interactions with cytosolic proteins.

We determined the solution structure of the EphB2 receptor SAM domain and studied its association behavior. The structure consists of five helices forming a compact structure without binding pockets or exposed conserved aromatic residues. Concentration-dependent chemical shift changes of NMR signals reveal two distinct well-separated areas on the domains' surface sensitive to the formation of homotypic oligomers in solution. These findings are supported by analytical ultracentrifugation studies. The conserved Tyr932, which was reported to be essential for the interaction with SH2 domains after phosphorylation, is buried in the hydrophobic core of the structure.

The weak capability of the isolated EphB2 receptor SAM domain to form oligomers is supposed to be relevant in vivo when the driving force of ligand binding induces receptor oligomerization. A formation of SAM tetramers is thought to provide an appropriate contact area for the binding of a low-molecular-weight phosphotyrosine phosphatase and to initiate further downstream responses.

The urea-induced equilibrium unfolding of the α subunit of tryptophan synthase (αTS), a single domain α/β barrel protein, displays a stable intermediate at ∼3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784–792). The same experiment, monitored by one-dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab-Rincón G et al., 1993, Biochemistry 32:13981–13990). To further test and quantify the implied four-state model, N [rlarr ] I1 [rlarr ] I2 [rlarr ] U, the urea-induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far-UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol−1, respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers ∼6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of αTS.

Following hints from X-ray data (Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547–10553; Yoshikawa S et al., 1998, Science 280:1723–1729), chemical evidence is presented from four distantly related cytochrome-c oxidases for the existence of a copperB-coordinated His240–Tyr244) cross-link at the O2-activating Heme Fea3–CuB center in the catalytic subunit I of the enzyme. The early evolutionary invention of this unusual structure may have prevented demaging [bull ]OH-radical release at e−-transfer to dioxygen and thus have enabled O2 respiration.

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 Å resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63–75) and to the corresponding domain in the 2.6 Å crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411–8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its α-β-α architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central β-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.

Two equilibrium intermediates have previously been observed in the urea denaturation of the α subunit of tryptophan synthase (αTS) from Escherichia coli, an eight-stranded β/α barrel protein. In the current study, a series of amino-terminal fragments were characterized to probe the elementary folding units that may be in part responsible for this complex behavior. Stop-codon mutagenesis was used to produce eight fragments ranging in size from 105–214 residues and containing incremental elements of secondary structure. Equilibrium studies by circular dichroism indicate that all of these fragments are capable of adopting secondary structure. All except for the shortest fragment fold cooperatively. The addition of the fourth, sixth, and eighth β-strands leads to distinct increases in structure, cooperativity, and/or stability, suggesting that folding involves the modular assembly of βαβ supersecondary structural elements. One-dimensional NMR titrations at high concentrations of urea, probing the environment around His92, were also performed to test for the presence of residual structure in the fragments. All fragments that contained the first four βα units of structure exhibited a cooperative unfolding transition at high concentrations of urea with significant but reduced stability relative to the full-length protein. These results suggest that the residual structure in αTS requires the participation of hydrophobic residues in multiple β-strands that span the entire sequence.