The ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS-1 recognize different DNA sequences with a core element centered over 5′-GGAA-3′ and 5′-GGAA/T-3′, respectively. To understand the molecular basis of this recognition, we carried out site-directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5′-GGAA-3′ and 5′-GGAT-3′ containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein-DNA interaction with the 5′-GGAA-3′ containing oligonucleotide caused by a faster release of the protein from the DNA (koff tenfold higher than the wild-type protein). With the double mutant A231S-N236Y, we obtained an increase in binding affinity and stability toward both 5′-GGAA-3′ and 5′-GGAT-3′ containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5′-GGAA-3′ and 5′-GGAT-3′ oligonucleotides. The free energy of dissociation for the double mutant A231S-N236Y with 5′-GGAA-3′ (ΔΔG((A231S-N236Y) − (N236Y)) = −1.2 kcal mol−1) confirm the stabilizing effect of this mutant in the protein-DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5′-GGAA-3′ and 5′-GGAT-3′ sequences, while A231S mutation modulates the degree of specificity toward 5′-GGAA-3′ and 5′GGAT-3′ sequences. This study explains why wild-type PU.1 does not recognize 5′-GGAT-3′ sequences and in addition broadens our understanding of 5′-GGAA/T-3′ recognition by ETS protein family members.

Tryptophan residues have been introduced into two domains of the human U1A protein to probe solution dynamics. The full length protein contains 282 residues, separated into three distinct domains: the N-terminal RBD1 (RNA Binding Domain I), consisting of amino acids 1–101; the C-terminal RBD2, residues 202–282; and the intervening linker region. Tryptophan residues have been substituted for specific phenylalanine residues on the surface of the β-sheet of either RBD1 or RBD2, thus introducing a single solvent exposed tryptophan as a fluorescence reporter. Both steady-state and time-resolved fluorescence measurements of the isolated RBD domains show that each tryptophan experiences a unique environment on the β-sheet surface. The spectral properties of each tryptophan in RBD1 and RBD2 are preserved in the context of the U1A protein, indicating these domains do not interact with each other or with the linker region. The rotational correlation times of the isolated RBDs and the whole U1A, determined by dynamic polarization measurements, show that the linker region is highly flexible such that each RBD exhibits uncorrelated motion.

N5-(l-1-carboxyethyl)-l-ornithine synthase [E.C. 1.5.1.24] (CEOS) from Lactococcus lactis has been cloned, expressed, and purified from Escherichia coli in quantities sufficient for characterization by biophysical methods. The NADPH-dependent enzyme is a homotetramer (Mr ≅ 140,000) and in the native state is stabilized by noncovalent interactions between the monomers. The far-ultraviolet circular dichroism spectrum shows that the folding pattern of the enzyme is typical of the α,β family of proteins. CEOS contains one tryptophan (Trp) and 19 tyrosines (Tyr) per monomer, and the fluorescence spectrum of the protein shows emission from both Trp and Tyr residues. Relative to N-acetyltyrosinamide, the Tyr quantum yield of the native enzyme is about 0.5. All 19 Tyr residues are titratable and, of these, two exhibit the uncommonly low pKa of ∼8.5, 11 have pKa ∼ 10.75, and the remaining six titrate with pKa ∼ 11.3. The two residues with pKa ∼ 8.5 contribute approximately 40% of the total tyrosine emission, implying a relative quantum yield >1, probably indicating Tyr-Tyr energy transfer. In the presence of NADPH, Tyr fluorescence is reduced by 40%, and Trp fluorescence is quenched completely. The latter result suggests that the single Trp residue is either at the active site, or in proximity to the sequence GSGNVA, that constitutes the βαβ fold of the nucleotide-binding domain. Chymotrypsin specifically cleaves native CEOS after Phe255. Although inactivated by this single-site cleavage of the subunit, the enzyme retains the capacity to bind NADPH and tetramer stability is maintained. Possible roles in catalysis for the chymotrypsin sensitive loop and for the low pKa Tyr residues are discussed.

The structural and dynamical behavior of the 41–56 β-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300 K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.

The assumption that the intrinsic α-helical propensities of the amino acids are position independent was critical in several helix/coil transition theories. In the first paper of these series, we reported that this is not the case for Gly and nonpolar aliphatic amino acids (Val, Leu, Met, and Ile). Here we have analyzed the helical intrinsic propensities of noncharged polar residues (Ser, Thr, Asn, and Gln) at different positions of a model polyalanine-based peptide. We found that Thr is more favorable (by ∼0.3 kcal/mol) at positions N1 and N2 than in the helix center, although for Ser, Asn, and Gln the differences are smaller (±0.2 kcal/mol), and in many cases within the experimental error. There is a reasonable agreement (±0.2 kcal/mol) between the calculated free energies, using the ECEPP/2 force field equipped with a hydration potential, and the experimental data, except at position N1.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 ± 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.

Low catalytic efficiency of basal-state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo-IRKD. Bound Mg-adenine nucleoside di- and triphosphates increased the cleavage rate with half-maximal effects observed at 0.4–0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino-terminal and carboxy-terminal core-flanking regions of the IRKD had no statistically significant impact on the ligand-dependent or -independent activation loop cleavages. Furthermore, the core-flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride-induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses ≥90% of the ligand-free basal state kinase. However, normal intracellular concentrations of Mg-adenine nucleotides, which are in the millimolar range, would favor a basal-state conformation of the activation loop that is more accessible.

Previous investigation has shown that at 22 °C and in the presence of the chaperonin GroEL, the slowest step in the refolding of Escherichia coli dihydrofolate reductase (EcDHFR) reflects release of a late folding intermediate from the cavity of GroEL (Clark AC, Frieden C, 1997, J Mol Biol 268:512–525). In this paper, we investigate the effects of potassium, magnesium, and MgADP on the release of the EcDHFR late folding intermediate from GroEL. The data demonstrate that GroEL consists of at least two conformational states, with apparent rate constants for EcDHFR release that differ by four- to fivefold. In the absence of potassium, magnesium, and ADP, ∼80–90% of GroEL resides in the form with the faster rate of release. Magnesium and potassium both shift the distribution of GroEL forms toward the form with the slower release rate, though cooperativity for the magnesium-induced transition is observed only in the presence of potassium. MgADP at low concentrations (0–50 μM) shifts the distribution of GroEL forms toward the form with the faster release rate, and this effect is also potassium dependent. Nearly identical results were obtained with a GroEL mutant that forms only a single ring, demonstrating that these effects occur within a single toroid of GroEL. In the presence of saturating magnesium, potassium, and MgADP, the apparent rate constant for the release of EcDHFR from wild-type GroEL at 22 °C reaches a limiting value of 0.014 s−1. For the single ring mutant of GroEL, the rate of EcDHFR release under the same conditions reaches a limiting value of 0.024 s−1, suggesting that inter-ring negative cooperativity exists for MgADP-induced substrate release. The data suggest that MgADP preferentially binds to one conformation of GroEL, that with the faster apparent rate constant for EcDHFR release, and induces a conformational change leading to more rapid release of substrate protein.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine-containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes.

GroEL minichaperones have potential in the biotechnology industry for the refolding of recombinant proteins. With the aim of enhancing and widening their use, we have created two highly stable functional variants of minichaperone GroEL(193–345). A sequence alignment of 130 members of the chaperonin 60 (Cpn60) family was used to design 37 single mutations. Two small-to-large mutations, A223T, A223V and one similar-size mutation, M233L, all located in the hydrophobic core were found to stabilize the protein by more than 1 kcal mol−1 each. Six stabilizing mutations were combined, yielding two multiple mutants that were 6.99 and 6.15 kcal mol−1 more stable than wild-type protein. Even though some of the substituted residue pairs are close to each other in the protein structure, the energetic effects of mutation are approximately additive. In particular, the stabilizing substitution A223T is unexpected and would have been missed by purely structural analysis. In the light of previously reported successes employing similar methods with several other proteins, our results show that a homology based approach is a simple and efficient method of increasing the stability of a protein.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 Å. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 Å). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 Å3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.

The human Theta class glutathione transferase GSTT2-2 has a novel sulfatase activity that is not dependent on the presence of a conserved hydrogen bond donor in the active site. Initial homology modeling and the crystallographic studies have identified three conserved Arg residues that contribute to the formation of (Arg107 and Arg239), and entry to (Arg242), a sulfate binding pocket. These residues have been individually mutated to Ala to investigate their potential role in substrate binding and catalysis. The mutation of Arg107 had a significant detrimental effect on the sulfatase reaction, while the Arg242 mutation caused only a small reduction in sulfatase activity. Surprisingly, the Arg239 had an increased activity resulting from a reduction in stability. Thus, Arg239 appears to play a role in maintaining the architecture of the active site. Electrostatic calculations performed on the wild-type and mutant forms of the enzyme are in good agreement with the experimental results. These findings, along with docking studies, suggest that prior to conjugation, the location of 1-menaphthyl sulfate, a model substrate for the sulfatase reaction, is approximately midway between the position ultimately occupied by the naphthalene ring of 1-menaphthylglutathione and the free sulfate. It is further proposed that the Arg residues in and around the sulfate binding pocket have a role in electrostatic substrate recognition.

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2–1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1–47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48–64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30–400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1–47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1–47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by β-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6–Cys14, Cys16–Cys28, Cys22–Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507–13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 μM).

For the past 24 years, high-resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been the technique of choice for analyzing the protein composition of cells, tissues, and fluids, as well as for studying changes in global patterns of gene expression elicited by a wide array of effectors. The technique, which was originally described independently by O'Farrell and Klose, offers the highest resolution of protein separation and, therefore, plays a central role in “proteomics,” an area of functional genomics that combines a plethora of techniques to resolve (2-D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting), quantify (scanners, phosphorimagers), and characterize proteins, as well as to store (image analysis and 2-D PAGE databases), communicate, and interface protein data with that emerging from the various genome mapping and sequencing projects. Proteomics, together with genomics, cDNA arrays, phage antibody libraries, and transgenics and knockouts, today provide an impressive array of technologies for studying gene expression in both health and disease.