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A M Gronenborn

Publications and source records attributed to A M Gronenborn.

At least 19 recordsLinked to original sources
PubMed2001

Structure and dynamics of MarA-DNA complexes: an NMR investigation.

An unanswered question regarding gene regulation is how certain proteins are capable of binding to DNA with high affinity at specific but highly degenerate consensus sequences. We have investigated the interactions between the Escherichia coli transcription factor, MarA, and its diverse binding sites using NMR techniques. Complete resonance assignments for the backbone of the MarA protein complexed with DNA oligomers corresponding to its binding sites at the mar, fumC, micF and the fpr promoters were obtained. Secondary structure analysis based on chemical shifts reveals that regions identified as helical in the X-ray structure of the MarA-mar complex are present in the solution structure, although some of the helices are less well defined. The chemical shift differences between the four complexes confirm that helix 3 and helix 6 constitute the major DNA-binding elements. However, in striking contrast with the X-ray data: (i) the protein appears to be present in two or more conformations in each of the complexes; (ii) no slowly exchanging N(zeta)H(2) protons (indicative of hydrogen bonded groups) were observed by NMR for the two arginine residues proposed to form crucial hydrogen bonds in the X-ray structure; and (iii) regions at the N terminus, not observed in the X-ray structure, may be involved in DNA-binding. Taken together, the NMR results indicate that MarA in its complexes with DNA target sites is in a highly dynamic state, allowing for small but significant rearrangements of the side-chains and/or backbone to bind to the different DNA sequences.

Bacterial Proteins↗
PubMed2001

Folded monomer of HIV-1 protease.

The mature human immunodeficiency virus type 1 protease rapidly folds into an enzymatically active stable dimer, exhibiting an intricate interplay between structure formation and dimerization. We now show by NMR and sedimentation equilibrium studies that a mutant protease containing the R87K substitution (PR(R87K)) within the highly conserved Gly(86)-Arg(87)-Asn(88) sequence forms a monomer with a fold similar to a single subunit of the dimer. However, binding of the inhibitor DMP323 to PR(R87K) produces a stable dimer complex. Based on the crystal structure and our NMR results, we postulate that loss of specific interactions involving the side chain of Arg(87) destabilizes PR(R87K) by perturbing the inner C-terminal beta-sheet (residues 96-99 from each monomer), a region that is sandwiched between the two beta-strands formed by the N-terminal residues (residues 1-4) in the mature protease. We systematically examined the folding, dimerization, and catalytic activities of mutant proteases comprising deletions of either one of the terminal regions (residues 1-4 or 96-99) or both. Although both N- and C-terminal beta-strands were found to contribute to dimer stability, our results indicate that the inner C-terminal strands are absolutely essential for dimer formation. Knowledge of the monomer fold and regions critical for dimerization may aid in the rational design of novel inhibitors of the protease to overcome the problem of drug resistance.

Amino Acid Sequence↗
PubMed2001

Probing the structure and stability of a hybrid protein: the human-E. coli thioredoxin chimera.

The structure and stability of a hybrid protein composed of N-terminal human and C-terminal E. coli thioredoxin domains were investigated by NMR, fluorescence, and circular dichroism spectroscopy. We demonstrate that the chimeric protein is correctly folded and exhibits the common thioredoxin architecture. However, the stability of the hybrid protein toward thermal and chemical denaturation is clearly reduced when compared with both parent proteins. Altogether, our data indicate that the interface between the two folding units of thioredoxin is tolerant toward changes in exact interdigitation of side chains, allowing for the formation of the unique overall thioredoxin fold. Further, the gene encoding the human-E. coli chimera was tested in vivo whether it supports the assembly of filamentous phages. No complementation of a thioredoxin-deficient E. coli mutant for the replication of the phages M13 or fd was observed, suggesting that parts of the overall protein structure in the N-terminal domain are crucial for this activity.

Amino Acid Sequence↗
PubMed2001

Solution structure, backbone dynamics and chitin binding of the anti-fungal protein from Streptomyces tendae TU901.

AFP1 is a recently discovered anti-fungal, chitin-binding protein from Streptomyces tendae Tü901. Mature AFP1 comprises 86 residues and exhibits limited sequence similarity to the cellulose-binding domains of bacterial cellulases and xylanases. No similarity to the Cys and Gly-rich domains of plant chitin-binding proteins (e.g. agglutinins, lectins, hevein) is observed. AFP1 is the first chitin-binding protein from a bacterium for which anti-fungal activity was shown. Here, we report the three-dimensional solution structure of AFP1, determined by nuclear magnetic resonance spectroscopy. The protein contains two antiparallel beta-sheets (five and four beta-strands each), that pack against each other in a parallel beta-sandwich. This type of architecture is conserved in the functionally related family II of cellulose-binding domains, albeit with different connectivity. A similar fold is also observed in other unrelated proteins (spore coat protein from Myxococcus xanthus, beta-B2 and gamma-B crystallins from Bos taurus, canavalin from Jack bean). AFP1 is therefore classified as a new member of the betagamma-crystallin superfamily. The dynamics of the protein was characterized by NMR using amide 15N relaxation and solvent exchange data. We demonstrate that the protein exhibits an axially symmetric (oblate-like) rotational diffusion tensor whose principal axis coincides to within 15 degrees with that of the inertial tensor. After completion of the present structure of AFP1, an identical fold was reported for a Streptomyces killer toxin-like protein. Based on sequence comparisons and clustering of conserved residues on the protein surface for different cellulose and chitin-binding proteins, we postulate a putative sugar-binding site for AFP1. The inability of the protein to bind short chitin fragments suggests that certain particular architectural features of the solid chitin surface are crucial for the interaction.

Amino Acid Sequence↗
PubMed2001

Characterization of the cholesteric phase of filamentous bacteriophage fd for molecular alignment.

Residual dipolar couplings arise from small degrees of alignment of molecules in a magnetic field and have proven to provide valuable structural information. Colloidal suspensions of rod-shaped viruses and bacteriophages constitute a frequently employed medium for imparting such alignment onto biomolecules. The stability and behavior of the liquid crystalline phases with respect to solution conditions such as pH, ionic strength, and temperature vary, and characterization should benefit practical applications as well as theoretical understanding. In this Communication we describe the pH dependence of the cholesteric liquid crystalline phase of the filamentous bacteriophage fd and demonstrate that the alignment tensor of the solute protein is modulated by pH. We also report the interesting observation that the relative sign of the residual dipolar coupling changes at low pH values. In addition, we demonstrate that the degree of alignment inversely scales with the lengths of the phage particles for phages with identical mass and charge per unit length.

DNA, Bacterial↗
PubMed2000

Multisite fluorescence in proteins with multiple tryptophan residues. Apomyoglobin natural variants and site-directed mutants.

Time-resolved fluorescence experiments were carried out on a variety of apomyoglobins with one or two tryptophan (Trp) residues located at invariant positions 7 and 14 in the primary sequence. In all cases, the Trp fluorescence kinetics were resolved adequately into two discrete lifetime domains, and decay-associated spectra (DAS) were obtained for each decay component. The DAS resolved for unfolded proteins were indistinguishable by position of the emission maxima and the spectral shapes. The folded proteins revealed noticeable differences in the DAS, which relate to the diverse local environments around the Trp residues in the individual proteins. Furthermore, the DAS of wild-type protein possessing two Trp residues were simulated well by that of one Trp mutants either in the native, molten globule, or unfolded states. Overall, employing Trp fluorescence and site-directed mutagenesis allowed us to highlight the conformational changes induced by the single amino acid replacement and generate novel structural information on equilibrium folding intermediates. Specifically, it was found that conformational fluctuations in the local cluster around the evolutionarily conserved Trp(14) are very similar in the native and molten globule states of apomyoglobins. This result indicates that residues in the E and B helices contributing to this cluster are most likely involved in the stabilization of the overall architecture of the structured molten globule intermediate.

Amino Acid Sequence↗
PubMed2000

Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1.

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.

Antigens, Bacterial↗
PubMed2000

Characterization of surfactant liquid crystal phases suitable for molecular alignment and measurement of dipolar couplings.

Media employed for imparting partial alignment onto solute molecules have recently attracted considerable attention, since they permit the measurement of NMR parameters for solute biomolecules commonly associated with solid state NMR. Here we characterize a medium which is based on a quasi-ternary surfactant system comprising cetylpyridinium bromide/hexanol/sodium bromide. We demonstrate that dilute solutions of this system can exist in liquid crystalline phases which orient in the magnetic field and allow the measurement of residual dipolar couplings under a variety of conditions. The present system is extremely versatile and robust, tolerating different buffer conditions, temperature ranges and concentrations.

Bromides↗
PubMed2000

Pressure alters electronic orbital overlap in hydrogen bonds.

Pressure-induced changes in 3hJ(NC') scalar couplings through hydrogen bonds were investigated in the immunoglobulin binding domain of streptococcal protein G. 1H, 15N and 13C triple-resonance NMR spectroscopy coupled with the on-line high pressure cell technique was used to monitor 3hJ(NC') scalar couplings at 30 and 2000 bar in uniformly labeled 15N and 13C protein isotopes. Both increased and decreased 3hJ(NC') scalar couplings were observed at high pressure. No correlation with secondary structure was apparent. The difference in coupling constants as well as pressure-induced chemical shift data suggests a compaction of the helix ends and an increase of the helix pitch at its center in response to pressure. Our data provides the first direct evidence that the electronic orbital overlap in protein backbone hydrogen bonds is altered by pressure.

Bacterial Proteins↗
PubMed2000

31P NMR analysis of the DNA conformation induced by protein binding SRY/DNA complexes.

Complexes of the HMG box protein SRY with two duplexes of 8 and 14 base pairs have been studied by 31P NMR and complete assignment of all phosphorus signals of the bound DNA duplexes are presented. While for the free DNA, all 31P signals display limited spectral dispersion (< 0.8 p.p.m.) for the bound duplexes, 31P resonances are spread over 2 p.p.m. Based on the previously published 3D structure of hSRY-HMG, with the 8 mer it is demonstrated that the upfield shifted resonances correspond to the site of partial intercalation of an isoleucine side chain into the DNA. Moreover, the observation of significant difference in linewidths between the two duplexes allows to estimate lifetime of the complexes from 31P-31P 2D exchange experiments.

DNA↗
PubMed1999

Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping.

The crystal structure of cyanovirin-N (CV-N), a protein with potent antiviral activity, was solved at 1.5 A resolution by molecular replacement using as the search model the solution structure previously determined by NMR. The crystals belong to the space group P3221 with one monomer of CV-N in each asymmetric unit. The primary structure of CV-N contains 101 residues organized in two domains, A (residues 1 to 50) and B (residues 51 to 101), with a high degree of internal sequence and structural similarity. We found that under the conditions of the crystallographic experiments (low pH and 26 % isopropanol), two symmetrically related monomers form a dimer by domain swapping, such that domain A of one monomer interacts with domain B' of its crystallographic symmetry mate and vice versa. Because the two swapped domains are distant from each other, domain swapping does not result in additional intramolecular interactions. Even though one of the protein sample solutions that was used for crystallization clearly contained 100 % monomeric CV-N molecules, as judged by various methods, we were only able to obtain crystals containing domain-swapped dimers. With the exception of the unexpected phenomenon of domain swapping, the crystal structure of CV-N is very similar to the NMR structure, with a root-mean-square deviation of 0.55 A for the main-chain atoms, the best agreement reported to date for structures solved using both techniques.

Antiviral Agents↗
PubMed1999

Disordered water within a hydrophobic protein cavity visualized by x-ray crystallography.

Water in the hydrophobic cavity of human interleukin 1beta, which was detected by NMR spectroscopy but was invisible by high resolution x-ray crystallography, has been mapped quantitatively by measurement and phasing of all of the low resolution x-ray diffraction data from a single crystal. Phases for the low resolution data were refined by iterative density modification of an initial flat solvent model outside the envelope of the atomic model. The refinement was restrained by the condition that the map of the difference between the electron density distribution in the full unit cell and that of the atomic model be flat within the envelope of the well ordered protein structure. Care was taken to avoid overfitting the diffraction data by maintaining phases for the high resolution data from the atomic model and by a resolution-dependent damping of the structure factor differences between data and model. The cavity region in the protein could accommodate up to four water molecules. The refined solvent difference map indicates that there are about two water molecules in the cavity region. This map is compatible with an atomic model of the water distribution refined by using XPLOR. About 70% of the time, there appears to be a water dimer in the central hydrophobic cavity, which is connected to the outside by two constricted channels occupied by single water molecules approximately 40% of the time on one side and approximately 10% on the other.

Computer Simulation↗
PubMed1999

Autoprocessing of HIV-1 protease is tightly coupled to protein folding.

In the Gag-Pol polyprotein of HIV-1, the 99-amino acid protease is flanked at its N-terminus by a transframe region (TFR) composed of the transframe octapeptide (TFP) and 48 amino acids of the p6pol, separated by a protease cleavage site. The intact precursor (TFP-p6pol-PR) has very low dimer stability relative to that of the mature enzyme and exhibits negligible levels of stable tertiary structure. Thus, the TFR functions by destabilizing the native structure, unlike proregions found in zymogen forms of monomeric aspartic proteases. Cleavage at the p6pol-PR site to release a free N-terminus of protease is concomitant with the appearance of enzymatic activity and formation of a stable tertiary structure that is characteristic of the mature protease as demonstrated by nuclear magnetic resonance. The release of the mature protease from the precursor can either occur in two steps at pH values of 4 to 6 or in a single step above pH 6. The mature protease forms a dimer through a four-stranded beta-sheet at the interface. Residues 1-4 of the mature protease from each subunit constitute the outer strands of the beta-sheet, and are essential for maintaining the stability of the free protease but are not a prerequisite for the formation of tertiary structure and catalytic activity. Our experimental results provide the basis for the model proposed here for the regulation of the HIV-1 protease in the viral replication cycle.

Amino Acid Sequence↗
PubMed1999

Solution structure of the 40,000 Mr phosphoryl transfer complex between the N-terminal domain of enzyme I and HPr.

The solution structure of the first protein-protein complex of the bacterial phosphoenolpyruvate: sugar phosphotransferase system between the N-terminal domain of enzyme I (EIN) and the histidine-containing phosphocarrier protein HPr has been determined by NMR spectroscopy, including the use of residual dipolar couplings that provide long-range structural information. The complex between EIN and HPr is a classical example of surface complementarity, involving an essentially all helical interface, comprising helices 2, 2', 3 and 4 of the alpha-subdomain of EIN and helices 1 and 2 of HPr, that requires virtually no changes in conformation of the components relative to that in their respective free states. The specificity of the complex is dependent on the correct placement of both van der Waals and electrostatic contacts. The transition state can be formed with minimal changes in overall conformation, and is stabilized in favor of phosphorylated HPr, thereby accounting for the directionality of phosphoryl transfer.

Amino Acid Sequence↗
PubMed1999

Is human thioredoxin monomeric or dimeric?

We have examined the molecular weight and rotational correlation time of human thioredoxin by analytical ultracentrifugation and NMR spectroscopy, respectively. Two variants of human thioredoxin were studied, namely human thioredoxin identical in amino acid sequence to the one whose NMR structure we previously determined (C62A, C69A, C73A, M74T) and human thioredoxin (C62A, C69A, C73A, M74) containing the wild-type amino acid methionine at position 74. In both cases, the experimental data indicate that the predominant species is monomeric and we find no evidence for the existence of a well-defined dimeric form as was observed in the recently reported crystal structure (Weichsel et al., 1996) of human thioredoxin and the C73S mutant.

Humans↗