Biomedical subjects

F W Dahlquist

Publications and source records attributed to F W Dahlquist.

At least 19 recordsLinked to original sources

PubMed2001

Thermodynamic basis for the increased thermostability of CheY from the hyperthermophile Thermotoga maritima.

The CheY protein isolated from the hyperthermophile Thermotoga maritima is much more resistant to thermally induced unfolding than is its counterpart from the mesophile Bacillus subtilis. To determine the basis of this increased thermostability, the temperature dependence of the free energy of unfolding was determined for these CheY homologues using denaturant-induced unfolding experiments. This allowed comparison of T. maritima CheY with B. subtilis CheY and determination of the thermodynamic qualities responsible for the enhanced thermostability of T. maritima CheY. The stability of the thermophilic CheY protein is a direct result of the increased enthalpy contribution at the temperature of zero entropy, T(s), and the decreased heat capacity change upon unfolding, resulting in a decreased dependence of the free energy of unfolding on temperature. It was found that neither purely entropic nor purely enthalpic contributions alone (as reflected by T(s)) were sufficient to account for the increase in stability.

Amino Acid Sequence↗

PubMed2001

A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C.

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4.10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the 1H NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine Hdelta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pKa of 8.0 at 6 degrees C, which is identified with the pKa of His32. The Hdelta1 signal is modulated by rapid exchange of the Hepsilon2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 A at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor > or = 100.

Bacillus cereus↗

PubMed2001

Probing slow time scale dynamics at methyl-containing side chains in proteins by relaxation dispersion NMR measurements: application to methionine residues in a cavity mutant of T4 lysozyme.

A relaxation dispersion-based NMR experiment is presented for the measurement and quantitation of micros-ms dynamic processes at methyl side-chain positions in proteins. The experiment measures the exchange contribution to the 13C line widths of methyl groups using a constant-time CPMG scheme. The effects of cross-correlated spin relaxation between dipole-dipole and dipole-CSA interactions as well as the effects of scalar coupling responsible for mixing of magnetization modes during the course of the experiment have been investigated in detail both theoretically and through simulations. It is shown that the complex relaxation properties of the methyl spin system do not complicate extraction of accurate exchange parameters as long as care is taken to ensure that appropriate magnetization modes are interchanged in the middle of the constant-time CPMG period. An application involving the measurement of relaxation dispersion profiles of methionine residues in a Leu99Ala substitution of T4 lysozyme is presented. All of the methionine residues are sensitive to an exchange event with a rate on the order of 1200 s(-1) at 20 degrees C that may be linked to a process in which hydrophobic ligands are able to rapidly bind to the cavity that is present in this mutant.

Algorithms↗

PubMed2001

What is the average conformation of bacteriophage T4 lysozyme in solution? A domain orientation study using dipolar couplings measured by solution NMR.

Lysozyme from T4 bacteriophage is comprised of two domains that are both involved in binding substrate. Although wild-type lysozyme has been exclusively crystallized in a closed form that is similar to the peptidoglycan-bound conformation, a more open structure is thought to be required for ligand binding. To determine the relative arrangement of domains within T4 lysozyme in the solution state, dipolar couplings were measured in several different dilute liquid crystalline media by solution NMR methods. The dipolar coupling data were analyzed with a domain orientation procedure described previously that utilizes high- resolution X-ray structures. The cleft between the domains is significantly larger in the average solution structure than what is observed in the X-ray structure of the ligand-free form of the protein (approximately 17 degrees closure from solution to X-ray structures). A comparison of the solution domain orientation with X-ray-derived structures in the protein data base shows that the solution structure resembles a crystal structure obtained for the M6I mutant. Dipolar couplings were also measured on the lysozyme mutant T21C/T142C, which was oxidized to form an inter-domain disulfide bond (T4SS). In this case, the inter-domain solution structure was found to be more closed than was observed in the crystal (approximately 11 degrees). Direct refinement of lysozyme crystal structures with the measured dipolar couplings using the program CNS, establishes that this degree of closure can be accommodated whilst maintaining the inter-domain cystine bond. The differences between the average solution conformations obtained using dipolar couplings and the crystal conformations for both forms of lysozyme investigated in this study illustrate the impact that crystal packing interactions can have on the arrangement of domains within proteins and the importance of alternative methods to X-ray crystallography for evaluating inter-domain structure.

Bacteriophage T4↗

PubMed2001

Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme.

A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.

Asparagine↗

PubMed2001

Measurement of one-bond 1H-13C, couplings in backbone-labelled proteins.

NMR dipole-dipole couplings between protein backbone nuclei (1H(alpha), 13C(alpha), 15N, 1H(N), 13C') offer enormous scope for the rapid determination of protein global folds. Here, we show that measurement of one-bond splittings in the protein backbone is facilitated by use of protein that is selectively isotopically enriched only in the backbone atoms. In particular, 1H(alpha)-13C(alpha) couplings can be measured simply and with high sensitivity by use of conventional heteronuclear single quantum correlation (HSQC) techniques.

Amino Acids↗

PubMed2001

Studying excited states of proteins by NMR spectroscopy.

Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly. We have used novel relaxation dispersion NMR techniques to kinetically and thermodynamically characterize a transition between a highly populated (97%, 25 degrees C) ground state conformation and an excited state that is 2.0 kcal mol(-1) higher in free energy. A temperature-dependent study of the rates of interconversion between ground and excited states allows the separation of the free energy change into enthalpic (Delta H = 7.1 kcal mol(-1)) and entropic (T Delta S = 5.1 kcal mol(-1), 25 degrees C) components. The residues involved cluster about the cavity, providing evidence that the excited state facilitates ligand entry.

Amino Acid Substitution↗

PubMed2000

Flexibility and ligand exchange in a buried cavity mutant of T4 lysozyme studied by multinuclear NMR.

The Leu99-->Ala mutant of T4 lysozyme contains a large internal cavity in the core of its C-terminal domain that is capable of reversibly binding small hydrophobic compounds. Although the cavity is completely buried, molecules such as benzene or xenon can exchange rapidly in and out. The dynamics of the unliganded protein have been compared to the wild-type protein by measuring the NMR spin relaxation rates of backbone amide and side chain methyl nuclei. Many residues surrounding the cavity were found to be affected by a chemical exchange process with a rate of 1500 +/- 200 s(-1), which is quenched upon addition of saturating amounts of the ligand xenon. The relationship between the structure, dynamics, and energetics of the T4 lysozyme mutant is discussed.

Alanine↗

PubMed2000

Cooperative ordering in homeodomain-DNA recognition: solution structure and dynamics of the MATa1 homeodomain.

The mating type homeodomain proteins, MATa1 and MATalpha2, combine to form a heterodimer to bind DNA in diploid yeast cells. The a1-alpha2 heterodimer tightly and specifically binds haploid-specific gene operators to repress transcription. On its own, however, the a1 homeodomain does not bind DNA in a sequence-specific manner. To help understand this interaction, we describe the solution structure and backbone dynamics of the free a1 homeodomain. Free a1 in solution is an ensemble of structures having flexible hinges at the two turns in the small protein fold. Conformational changes in the a1 homeodomain upon ternary complex formation are located in the loop between helix 1 and helix 2, where the C-terminal tail of alpha2 binds to form the heterodimer, and at the C-terminus of helix 3, the DNA recognition helix. The observed differences, comparing the free and bound a1 structures, suggest a mechanism linking van der Waals stacking changes to the ordering of a final turn in the DNA-binding helix of a1. The tail of alpha2 induces changes in loop 1 of a1 that push it toward a properly folded DNA binding conformation.

Amino Acid Sequence↗

PubMed2000

Trans-substitution of the proximal hydrogen bond in myoglobin: I. Structural consequences of hydrogen bond deletion.

The structural role of a side-chain to side-chain protein hydrogen bond is examined using trans-substitution of the proximal histidine of myoglobin with methylimidazoles (Barrick, Biochemistry 1994;33:6546-6554). Modification of the chemical structure of exogenous ligands allows this hydrogen bond to be disrupted. Comparison of the crystal structures of H93G myoglobin complexed 4-methylimidazole (4meimd; methylation at carbon 4) and 1-methylimidazole (1meimd; methylation at the adjacent nitrogen, preventing hydrogen bonding between the imidazole ligand and the protein) shows that the polypeptide, heme, and methylimidazole orientations are the same within error. For 4meimd there appear to be major and minor conformations corresponding to different tautomeric states of the ligand. Conformational heterogeneity is also seen in the hyperfine-shifted region of the NMR spectrum of 4meimd complexed with high-spin H93G deoxyMb. The major conformation of the 4meimd ligand and the 1meimd ligand, as seen in the respective crystal structures, are quite similar except that the proximal ligand NH-to-Ser92-OH hydrogen bond is eliminated in the 1meimd complex, and instead the proximal ligand CH is adjacent to the Ser92-OH. Thus, this system provides a means to eliminate the Mb proximal hydrogen bond in a chemically and structurally conservative way.

Animals↗

PubMed2000

The 1.9 A resolution crystal structure of phosphono-CheY, an analogue of the active form of the response regulator, CheY.

To structurally characterize the activated state of the transiently phosphorylated signal transduction protein CheY, we have constructed an alpha-thiophosphonate derivative of the CheY D57C point mutant and determined its three-dimensional structure at 1.85 A resolution. We have also characterized this analogue with high-resolution NMR and studied its binding to a peptide derived from FliM, CheY's target component of the flagellar motor. The chemically modified derivative, phosphono-CheY, exhibits many of the chemical properties of phosphorylated wild-type CheY, except that it is indefinitely stable. Electron density for the alpha-thiophosphonate substitution is clear and readily interpretable; omit refinement density at the phosphorus atom is greater than 10sigma. The molecule shows a number of localized conformational changes that are believed to constitute the postphosphorylation activation events. The most obvious of these changes include movement of the side chain of the active site base, Lys 109, and a predominately buried conformation of the side chain of Tyr 106. In addition, there are a number of more subtle changes more distant from the active site involving the alpha4 and alpha5 helices. These results are consistent with our previous structural interpretations of other CheY activation mutants, and with our earlier hypotheses concerning CheY activation through propagation of structural changes away from the active site.

Bacterial Proteins↗

PubMed2000

Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme.

Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 +/- 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol.

Bacteriophage T4↗

PubMed1999

Methionine and alanine substitutions show that the formation of wild-type-like structure in the carboxy-terminal domain of T4 lysozyme is a rate-limiting step in folding.

In an attempt to identify a systematic relation between the structure of a protein and its folding kinetics, the rate of folding was determined for 20 mutants of T4 lysozyme in which a bulky, buried, nonpolar wild-type residue (Leu, Ile, Phe, Val, or Met) was substituted with alanine. Methionine, which approximated the size of the original side chain but which is of different shape and flexibility, was also substituted at most of the same sites. Mutations that substantially destabilize the protein and are located in the carboxy-terminal domain generally slow the rate of folding. Destabilizing mutations in the amino-terminal domain, however, have little effect on the rate of folding. Mutations that have little effect on stability tend to have little effect on the rate, no matter where they are located. These results suggest that, at the rate-limiting step, elements of structure in the C-terminal domain are formed and have a structure similar to that of the fully folded protein. Consistent with this, two variants that somewhat increase the rate of folding (Phe104 --> Met and Val149 --> Met) are located within the carboxy-terminal domain and maintain or improve packing with very little perturbation of the wild-type structure.

Alanine↗

PubMed1999

Identification of the binding interfaces on CheY for two of its targets, the phosphatase CheZ and the flagellar switch protein fliM.

CheY is the response regulator protein serving as a phosphorylation-dependent switch in the bacterial chemotaxis signal transduction pathway. CheY has a number of proteins with which it interacts during the course of the signal transduction pathway. In the phosphorylated state, it interacts strongly with the phosphatase CheZ, and also the components of the flagellar motor switch complex, specifically with FliM. Previous work has characterized peptides consisting of small regions of CheZ and FliM which interact specifically with CheY. We have quantitatively measured the binding of these peptides to both unphosphorylated and phosphorylated CheY using fluorescence spectroscopy. There is a significant enhancement of the binding of these peptides to the phosphorylated form of CheY, suggesting that these peptides share much of the binding specificity of the intact targets of the phosphorylated form of CheY. We also have used modern nuclear magnetic resonance methods to characterize the sites of interaction of these peptides on CheY. We have found that the binding sites are overlapping and primarily consist of residues in the C-terminal portion of CheY. Both peptides affect the resonances of residues at the active site, indicating that the peptides may either bind directly at the active site or exert conformational influences that reach to the active site. The binding sites for the CheZ and FliM peptides also overlap with the previously characterized CheA binding interface. These results suggest that interaction with these three proteins of the signal transduction pathway are mutually exclusive. In addition, since these three proteins are sensitive to the phosphorylation state of CheY, it may be that the C-terminal region of CheY is most sensitive for the conformational changes occurring upon phosphorylation.

Amino Acid Sequence↗

PubMed1999

The energetics of T4 lysozyme reveal a hierarchy of conformations.

We have used native state exchange to examine the energy landscape of the well-characterized protein T4 lysozyme. Although the protein exhibits two-state behavior by traditional probes, the energy landscape determined here is much more complex. The average stability of the C-terminal subdomain is significantly higher than that for the N-terminus suggesting at least two regions of unfolding. At a more detailed level, there appears to be a broad continuum of stabilities throughout each region. The overall subdomain hierarchy of energies does not mirror data on the folding pathway for this protein, challenging the relationship between energy landscapes and folding trajectories.

Bacteriophage T4↗

PubMed1999

Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution.

Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution.

Amino Acid Sequence↗

PubMed1998

Regulation of phosphatase activity in bacterial chemotaxis.

Bacterial chemotaxis is the most studied model system for signaling by the widely spread family of two-component regulatory systems. It is controlled by changes in the phosphorylation level of the chemotactic response regulator, CheY, mediated by a histidine kinase (CheA) and a specific phosphatase (CheZ). While it is known that CheA activity is regulated, via the receptors, by chemotactic stimuli, the input that may regulate CheY dephosphorylation by CheZ has not been found. We measured, by using stopped-flow fluorometry, the kinetics of CheZ-mediated dephosphorylation of CheY. The onset of dephosphorylation was delayed by approximately 50 ms after mixing phosphorylated CheY (CheY approximately P) with CheZ, and a distinct overshoot was observed in the approach to the new steady state of CheY approximately P. The delay and overshoot were not observed in a hyperactive mutant CheZ protein (CheZ54RC) that does not support chemotaxis in vivo and appears to be constitutively active. CheZ activity was cooperative with respect to CheY approximately P, with a Hill-coefficient of 2.5. The observed delayed modulation of CheZ activity and its cooperativity suggest that the phosphatase activity is regulated at the level of CheY approximately P-CheZ interaction. This novel kind of interplay between a response regulator and its phosphatase may be involved in signal tuning and in adaptation to chemotactic signals.

Bacterial Proteins↗

PubMed1998

Synthesis and biochemical characterization of an analogue of CheY-phosphate, a signal transduction protein in bacterial chemotaxis.

CheY is a signal transduction protein of the bacterial chemotaxis system that acts as a molecular switch to alter the swimming behavior of the bacterium. When CheY becomes phosphorylated at Asp57, CheY-Pi interacts with flagellar motor proteins, including FliM, to increase the likelihood that the flagellar motor will change its sense of rotation, increasing the frequency of tumbling. The structure of CheY in its dephosphorylated (inactive) state has been intensively investigated. The short lifetime ( approximately 20 s) of the aspartyl phosphate has precluded the complete structural determination of CheY-Pi. We have synthesized an analogue of CheY-Pi by alkylating an aspartate-to-cysteine mutant at position 57 of CheY to add a phosphonomethyl group at Cys57. This analogue, phosphono-CheY, is stable for months. Phosphono-CheY binds to two of the targets of CheY-Pi, FliM and CheZ, in a manner similar to that of CheY-Pi and much better than either unphosphorylated CheY or the unmodified form of D57C CheY. Phosphono-CheY also binds Mg(II) with a dissociation constant of approximately 6 mM at neutral pH and moderate salt level. These observations indicate that phosphono-CheY is a good biochemical analogue of CheY-Pi.

Alkylation↗