Biomedical subjects

F W Dahlquist

Publications and source records attributed to F W Dahlquist.

At least 55 records · Page 3Linked to original sources

PubMed1995

1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR.

Spo0F, sporulation stage 0 F protein, a 124-residue protein responsible, in part, for regulating the transition of Bacillus subtilis from a vegetative state to a dormant endospore, has been studied by high-resolution NMR. The 1H, 15N, and 13C chemical shift assignments for the backbone residues have been determined from analyses of 3D spectra, 15N TOCSY-HSQC, 15N NOESY-HSQC, HNCA, and HN(CO)CA. Assignments for many sidechain proton resonances are also reported. The secondary structure, inferred from short- and medium-range NOEs, 3JHN alpha coupling constants, and hydrogen exchange patterns, define a topology consistent with a doubly wound (alpha/beta)5 fold. Interestingly, comparison of the secondary structure of Spo0F to the structure of the Escherichia coli response regulator, chemotaxis Y protein (CheY) (Volz K, Matsumura P, 1991, J Biol Chem 266:15511-15519; Bruix M et al., 1993, Eur J Biochem 215:573-585), show differences in the relative length of secondary structure elements that map onto a single face of the tertiary structure of CheY. This surface may define a region of binding specificity for response regulators. Magnesium titration of Spo0F, followed by amide chemical shift changes, gives an equilibrium dissociation constant of 20 +/- 5 mM. Amide resonances most perturbed by magnesium binding are near the putative site of phosphorylation, Asp 54.

Amino Acid Sequence↗

PubMed1995

15N, 13C, and 1H NMR assignments and secondary structure for T4-lysozyme.

Sequence-specific 1H, 13C, and 15N backbone assignments and 1H and 13C side-chain assignments have been identified for T4-lysozyme (164 residues, MW = 18.7 kDa). A variety of double- and triple-resonance 3D techniques were used. Some of these methods were applied in unconventional ways and a detailed description of the advantages and disadvantages of these approaches is given. Complete backbone resonances for 162 of the 164 residues and partial assignments for the remaining 2 residues were obtained. The 1H and 15N assignments are in agreement with those obtained previously by McIntosh et al. who used selective labeling (L.P. McIntosh et al., Biochemistry 29, 6341 (1990)). Complete proton and carbon side-chain assignments were made for 120 residues and partial side-chain assignments were made for 42 additional residues. A qualitative analysis of the medium-range NOESY data reveals a secondary structure consistent with the X-ray crystallographic structure.

Carbon Isotopes↗

PubMed1995

Localized perturbations in CheY structure monitored by NMR identify a CheA binding interface.

Phosphotransfer between the autophosphorylating histidine kinase CheA and the response regulator CheY represents a crucial step in the bacterial chemotaxis signal transduction pathway. The 15N-1H correlation spectrum of CheY complexed with an amino-terminal fragment of CheA exhibits specific localized differences in chemical shifts when compared to the spectrum of uncomplexed CheY. When mapped onto the three-dimensional structure of CheY, these changes define a region distinct from the active site. A single amino-acid substitution within this binding region on CheY, alanine to valine at position 103, significantly decreases the affinity of CheY for CheA. The binding face described by these changes partially overlaps a flagellar switch binding surface previously defined by mutagenesis.

Bacterial Proteins↗

PubMed1995

Structural features of the epsilon subunit of the Escherichia coli ATP synthase determined by NMR spectroscopy.

The tertiary fold of the epsilon subunit of the Escherichia coli F1F0 ATPsynthase (ECF1F0) has been determined by two- and three-dimensional heteronuclear (13C, 15N) NMR spectroscopy. The epsilon subunit exhibits a distinct two domain structure, with the N-terminal 84 residues of the protein forming a 10-stranded beta-structure, and with the C-terminal 48 amino acids arranged as two alpha-helices running antiparallel to one another (two helix hairpin). The beta-domain folds as a beta-sandwich with a hydrophobic interior between the two layers of the sandwich. The C-terminal two-helix hairpin folds back to the N-terminal domain and interacts with one side of the beta-domain. The arrangement of the epsilon subunit in the intact F1F0 ATP synthase involves interaction of the two helix hairpin with the F1 part, and binding of the open side of the beta-sandwich to the c subunits of the membrane-embedded F0 part.

Amino Acid Sequence↗

PubMed1994

Heterodimerization of the yeast homeodomain transcriptional regulators alpha 2 and a1: secondary structure determination of the a1 homeodomain and changes produced by alpha 2 interactions.

The homeodomain proteins, a1 and alpha 2, act cooperatively to regulate cell-type specific genes in yeast. The basis of this cooperativity is an interaction between the two proteins, forming a heterodimer that binds DNA tightly and specifically. A fragment containing the homeodomain of a1, a1(66-126), has been studied by NMR spectroscopy to gain secondary structure information and to characterize the changes in a1 upon heterodimerization with alpha 2. Heteronuclear (1H-15N) NMR methods were used to assign backbone resonances of the 61 amino acid fragment. The a1(66-126) secondary structure was determined using NOE connectivities, 3JHN alpha coupling constants and hydrogen exchange kinetic data. NMR data identify three helical segments separated by a loop and a tight turn that are the characteristic structural elements of homeodomain proteins. The a1 fragment was titrated with alpha 2(128-210), the homeodomain-containing fragment of alpha 2, to study changes in a1(66-126) spectra produced by alpha 2 binding. The a1(66-126) protein was labeled with 15N and selectively observed using isotope-edited NMR experiments. NMR spectra of bound a1(66-126) indicate that residues in helix 1, helix 2, and the loop connecting them are directly involved in the binding of the alpha 2 fragment. Relatively minor effects on the resonances from residues in helix 3, the putative DNA-binding helix, were noted upon alpha 2 binding. We have thus located a region of the a1 homeodomain important for specific protein recognition.

Amino Acid Sequence↗

PubMed1994

Signal transduction in chemotaxis. A propagating conformation change upon phosphorylation of CheY.

The CheY protein from Escherichia coli and Salmonella typhimurium are among the best characterized proteins of the receiver domain family of two component signal transduction systems in bacteria. Phosphorylation of CheY plays a central role in bacterial chemotaxis. However, it is not entirely clear how its state of phosphorylation contributes to its function. Genetic evidence suggests that CheY changes its conformation upon phosphorylation. We present evidence for this conformation change by comparing the NMR 15N-1H correlation spectra of CheY.Mg2+ complex and phospho-CheY in the presence of magnesium. Large changes in chemical shift are used as indicators of chemical changes and probable structural changes in the protein backbone. Our observations suggest that significant structural changes occur in CheY upon phosphorylation and that these changes are distinct from the changes produced by magnesium ion binding. In addition to residues Asn-59 and Gly-65 that are immediately adjacent to the site of phosphorylation at Asp-57, a large number of other residues show significant chemical shift changes as a result of phosphorylation. These include Met-17, Val-21, Asn-23, Gly-39, Met-60, Met-63, Asp-64, Leu-66, Glu-67, Leu-68, Leu-69, Met-85, Val-86, Thr-87, Ala-88, Asn-94, Val-107, Lys-109, Thr-112, Ala-113, Ala-114, and Asn-121. These results appear inconsistent with the recent suggestion that phosphorylation produces the same structural changes as magnesium binding (Bellsolell, L., Prieto, J., Serrano, L., and Coll, M. (1994) J. Mol. Biol. 238, 489-495). We find that some regions change overlap with a genetically defined motor binding face. We therefore propose that the conformation switch modulates the interaction of CheY with its target, the flagellar motor. Other regions also change, possibly reflecting the many different functions of CheY homologues.

Amino Acid Sequence↗

PubMed1994

Assignments, secondary structure, global fold, and dynamics of chemotaxis Y protein using three- and four-dimensional heteronuclear (13C,15N) NMR spectroscopy.

NMR spectroscopy has been used to study recombinant Escherichia coli CheY, a 128-residue protein involved in regulating bacterial chemotaxis. Heteronuclear three- and four-dimensional (3D and 4D) experiments have provided sequence-specific resonance assignments and quantitation of short-, medium-, and long-range distance restraints from nuclear Overhauser enhancement (NOE) intensities. These distance restraints were further supplemented with measurements of three-bond scalar coupling constants to define the local dihedral angles, and with the identification of amide protons undergoing slow solvent exchange from which hydrogen-bonding patterns were identified. The current model structure shows the same global fold of CheY as existing X-ray structures (Volz & Matsumura, 1991; Stock et al. 1993) with a (beta/alpha)5 motif of five parallel beta-strands at the central core surrounded by three alpha-helices on one face and with two on the opposite side. Heteronuclear 15N-1H relaxation experiments are interpreted to show portions of the protein structure in the Mg2+ binding loop are ill-defined because of slow motion (chemical exchange) on the NMR time scale. Moreover, the presence of Mg2+ disrupts the salt bridge between the highly conserved Lys-109 and Asp-57, the site of phosphorylation.

Amino Acid Sequence↗

PubMed1994

Heterodimerization of the yeast homeodomain transcriptional regulators alpha 2 and a1 induces an interfacial helix in alpha 2.

The homeodomain proteins a1 and alpha 2 act cooperatively to regulate cell type specific genes in yeast. The basis of the cooperativity is a weak interaction between the two proteins which forms heterodimers that bind DNA tightly and specifically. In this paper, we examine the mechanism of heterodimerization. We show that two relatively small fragments of a1 and alpha 2 are capable of heterodimerization and tight DNA binding. The alpha 2 fragment contains the homeodomain followed by the natural 22 C-terminal amino acids of the protein; these 22 amino acids are unstructured in the alpha 2 fragment. The a1 fragment contains only the homeodomain, indicating that the a1 homeodomain mediates both DNA binding and protein-protein interactions with alpha 2. We used isotope-edited NMR spectroscopy to study the interaction in solution of these two fragments. Samples in which only the alpha 2 fragment was uniformly labeled with 15N allowed us to visualize changes in the NMR spectra of the alpha 2 fragment produced by heterodimerization. We found that the a1 homeodomain perturbs the resonances of only the C-terminal tail of alpha 2; moreover, contact with a1 converts a portion of this tail (residues 193-203) from its unstructured state to an alpha-helix, as determined by J coupling and NOE measurements. Thus the heterodimerization of two homeodomain proteins involves the specific interaction between a tail of one protein and the homeodomain of the other. This interaction is accompanied by the acquisition of secondary structure in the tail.

Amino Acid Sequence↗

PubMed1994

Accommodation of amino acid insertions in an alpha-helix of T4 lysozyme. Structural and thermodynamic analysis.

One to four alanines were inserted by site-directed mutagenesis at three different locations within the alpha-helix comprising residues 39 to 50 in bacteriophage T4 lysozyme. All insertion mutants were correctly folded and catalytically active although the insertions led to a thermal destabilization by 1.1 to 4.2 kcal/mol when compared to wild-type. Variants that restored part of the loss in stability associated with the initial alanine insertions could be found by randomizing the inserted amino acids. In selected cases, directed mutagenesis of adjacent residues was also used to regain stability. Structural information obtained from X-ray crystallography and/or 2D-NMR for 10 different variants showed two distinct ways in which the protein responded to the amino acid insertions: (1) The inserted amino acids were incorporated into the helix by replacing preceding wild-type amino acids and causing a shift in register towards the N terminus. As a consequence, wild-type amino acids were translocated from the helix into the preceding loop. (2) Insertions caused a "looping out" within the alpha-helix. In this case the perturbation was confined to a minimal region in the immediate vicinity of the insertion. No change in the length of the helix was detected in either case. The structural response appears to be determined by the maintenance of the hydrophobic interface between the helix and the rest of the protein. This interface remains essentially intact in all variant structures. The results exemplify the plasticity and the adaptability of the protein structure which allows the incorporation of additional amino acids into a secondary structure element without large structural perturbations, as long as vital internal interactions are preserved. They also suggest that loops in proteins related by evolution can vary in length not only because of insertions within the loops themselves but also as a consequence of insertions within neighboring secondary structure elements.

Alanine↗

PubMed1993

Individual subunits of bacterial luciferase are molten globules and interact with molecular chaperones.

We have studied the assembly of a large heterodimeric protein, bacterial luciferase, by mixing purified subunits expressed separately in bacteria. The individual subunits alpha and beta contain much (66% and 50%, respectively) of the alpha-helix content of the native heterodimer as measured by circular dichroism, yet the alpha subunit lacks observable tertiary structure as measured by NMR. These results are consistent with the alpha subunit existing in a molten globule or collapsed form prior to assembly. The molecular chaperone GroEL binds reversibly to both subunits prior to assembly. Since these observations were obtained under physiological conditions, we propose that the molten globule exists as a stable form during folding or assembly in the cell. Either the molten globule form of the subunits is an authentic folding intermediate or it is in rapid equilibrium with one. GroEL may function by facilitating assembly through stabilization of these incompletely folded subunits.

Adenosine Triphosphate↗

PubMed1993

How amino-acid insertions are allowed in an alpha-helix of T4 lysozyme.

Studies of extant protein sequences indicate that amino-acid insertions and deletions are preferentially located in loop regions, which has traditionally been explained as the result of selection removing deleterious mutations within secondary structural elements from the population. But there is no a priori reason to discount the possibility that insertions within secondary structure could either be tolerated until compensatory mutations arise, or have effects that are propagated away from secondary structure into loops. Earlier studies have indicated that insertions are generally tolerated, although much less well within secondary structure elements than in loop regions. Here we show that amino-acid insertions in an alpha-helix of T4 lysozyme can be accepted in two different ways. In some cases the inserted amino acids are accommodated within the helix, leading to the translocation of wild-type residues from the helix to the preceding loop. In other cases the insertion causes a 'looping-out' in the first or last turn of the helix. The individual structural responses seem to be dominated by the maintenance of the interface between the helix and the rest of the protein.

Amino Acid Sequence↗

PubMed1992

Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway.

We examined the binding interactions of the methylation-dependent chemotaxis receptors Tsr and Tar with the chemotaxis-specific protein kinase CheA and the coupling factor CheW. Receptor directly bound CheW, but receptor-CheA binding was dependent upon the presence of CheW. These observations in combination with our previous identification of a CheW-CheA complex suggest that CheW physically links the kinase to the receptor. The ternary complex of receptor, CheW, and CheA is both kinetically and thermodynamically stable at physiological concentrations. Stability is not significantly altered by changes associated with attractant or repellent binding to the receptor. Such binding greatly modulates the kinase activity of CheA. Our results demonstrate that modulation of the kinase activity does not require association-dissociation of the ternary complex. This suggests that the receptor signal is transduced through conformational changes in the ternary complex rather than through changes in the association of the kinase CheA with receptor and/or CheW.

Bacterial Proteins↗

PubMed1992

Protein folding: assignment of the energetic changes of reversible chemical modifications to the folded or unfolded states.

Reversible chemical modifications of a series of single cysteine-containing variants of T4 lysozyme combined with thermal denaturation studies have been used to study the effects of these modifications on the stability of the protein. This allows dissection of the energetic effects of the modification on both the native and denatured states of this protein. At some sites modifications with various chemical reagents have essentially no effect on the stability of the protein, while at others, substantial changes in stability are observed. For example, chemical modification of cysteine at site 146 by cystamine (+NH3CH2CH2SSCH2-CH2NH3+) to form the mixed disulfide lowers the stability of the protein by about 1.1 kcal/mol. The reduction in the free energy of folding caused by the chemical modification is attributed to the destabilization of native state (0.9 kcal/mol), with only a relatively small effect from stabilization of the denatured state (0.2 kcal/mol). Chemical modifications of T4 lysozyme at site 146 with various chemical reagents show that the stability of the protein is lowered by a positively charged group and is relatively independent of the size of the side chains. This approach allows the investigation of the thermodynamic consequences of the reversible insertion of a wide variety of chemical entities at specific sites in proteins and, most importantly, allows dissection of the contribution of the chemical modifications to both the folded and unfolding states. It can be applied to almost any suitable macromolecular system.

Buffers↗

PubMed1992

Detection and characterization of an early folding intermediate of T4 lysozyme using pulsed hydrogen exchange and two-dimensional NMR.

Two-dimensional 1H-15N NMR techniques combined with pulsed hydrogen-deuterium exchange have been used to characterize the folding pathway of T4 lysozyme. In the unfolded state, there is little differential protection of the various amides from hydrogen exchange. In the native folded structure, 84 amides of the 164 residues are sufficiently spectrally resolved and protected from solvent exchange to serve as probes of the folding pathway. These probes are located in both the N-terminal and C-terminal domains of the native folded structure of the protein. The studies described here show that at least one intermediate is formed early during refolding at low denaturant concentrations. This intermediate (or intermediates) forms very rapidly (within the 10-ms temporal resolution of our mixing device) under the conditions used and is completed at least 10 times faster than the overall folding event. The intermediate(s) protect(s) from exchange a subset of amides in the N-terminal and C-terminal regions of the protein. In the final folded states these protected regions correspond to two alpha-helices and a beta-sheet region. These amides are protected from exchange by factors between 20 and 200 as compared to the fully unfolded protein. Protection of this magnitude is consistent with the formation of somewhat exposed secondary structure in these regions and could represent a "molten globule"-like or a "framework"-like structure for the intermediate(s) in which specific parts of the sequence form isolated secondary structures that are not stabilized by extensive tertiary interactions.

Amino Acid Sequence↗

PubMed1991

Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme.

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.

Amino Acid Sequence↗

PubMed1991

Sites of deamidation and methylation in Tsr, a bacterial chemotaxis sensory transducer.

The sensory transducer proteins in bacterial chemotaxis undergo two covalent modifications, deamidation and reversible methylation, in response to attractants and repellents. Oligonucleotide-directed mutagenesis was used to alter putative methylation and deamidation sites in one of the transducers to further define these sites and their role in chemotaxis. The mutations, in combination with peptide maps and Edman analysis, have clarified the sites of covalent modification in Tsr. Tsr contains six specific glutamates and glutamines that serve as methyl-accepting sites. An arginine-containing tryptic peptide (R1) has two sites, one at glutamate 493 and a newly located site at glutamate 502. A lysine-containing peptide (K1) has four methyl-accepting sites. Two of the lysine peptide sites are glutamates and can accept methyl groups without deamidation. The other two sites are glutamines and two methyl-accepting sites are created by two distinct deamidations. Both deamidations can occur on the same polypeptide chain. Single glutamate mutants have shown that one deamidation (at glutamine 311) proceeds rapidly, while the other deamidation (at glutamine 297) has a half-life of approximately 60 min under our experimental conditions.

Amides↗

PubMed1991

2H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer.

Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there.

Circular Dichroism↗

PubMed1991

Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA.

An essential step in the signal transduction pathway of Escherichia coli is the control of the protein kinase activity of CheA by the chemotaxis receptor proteins. This control requires the participation of the CheW protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCl/5 mM MgCl2, pH 7.4 at 4 degrees C) was determined to be in the physiologically relevant range of 17 microM. No evidence for cooperativity in the association of CheW with CheA was found.

Bacterial Proteins↗