Biomedical subjects

R James

Publications and source records attributed to R James.

At least 55 records · Page 3Linked to original sources

PubMed2000

Fish shoal composition: mechanisms and constraints.

Observations were made on three fish species (banded killifish (Fundulus diaphanus), golden shiner (Notemigonus crysoleucas) and white sucker (Catostomus commersoni)) in a temperate lake (New Brunswick, Canada) in order to investigate the relationship between shoal choice behaviour of individual fishes and shoal composition. Encounters between shoals were observed to take place every 1.1 min per shoal and an encounter lasted 3.7 s on average. The duration of shoal encounters was influenced by shoal size but not by differences between shoals in either body length or species. Conversely, the outcome of shoal encounters (i.e. ences between shoals in either body length or species. Conversely, the outcome of shoal encounters (i.e. whether or not an individual changes shoal) was influenced by body length and species differences but not by shoal size. Together, these results suggest that encounter duration itself is unlikely to have an important influence on encounter outcome. The collection of ten entire fish shoals showed that they were assorted by species and body length. A simulation model demonstrated that individual shoal choice behaviour alone could account for the generation and maintenance of the observed levels of size assortedness of shoals without invoking the existence of other sorting mechanisms such as differential swimming speeds. However, the generation of species assortedness was not predicted by the model. Furthermore, our data suggest that fish density acts as a constraint on shoal choice, influencing both shoal size and composition. This work has implications for studies on information transfer and reciprocal altruism within populations.

Animals↗

PubMed2000

Inhibition of a ribosome-inactivating ribonuclease: the crystal structure of the cytotoxic domain of colicin E3 in complex with its immunity protein.

BACKGROUND: The cytotoxicity of most ribonuclease E colicins towards Escherichia coli arises from their ability to specifically cleave between bases 1493 and 1494 of 16S ribosomal RNA. This activity is carried by the C-terminal domain of the colicin, an activity which if left unneutralised would lead to destruction of the producing cell. To combat this the host E. coli cell produces an inhibitor protein, the immunity protein, which forms a complex with the ribonuclease domain effectively suppressing its activity. RESULTS: We have solved the crystal structure of the cytotoxic domain of the ribonuclease colicin E3 in complex with its immunity protein, Im3. The structure of the ribonuclease domain, the first of its class, reveals a highly twisted central beta-sheet elaborated with a short N-terminal helix, the residues of which form a well-packed interface with the immunity protein. CONCLUSIONS: The structure of the ribonuclease domain of colicin E3 is novel and forms an interface with its inhibitor which is significantly different in character to that reported for the DNase colicin complexes with their immunity proteins. The structure also gives insight into the mode of action of this class of enzymatic colicins by allowing the identification of potentially catalytic residues. This in turn reveals that the inhibitor does not bind at the active site but rather at an adjacent site, leaving the catalytic centre exposed in a fashion similar to that observed for the DNase colicins. Thus, E. coli appears to have evolved similar methods for ensuring efficient inhibition of the potentially destructive effects of the two classes of enzymatic colicins.

Amino Acid Sequence↗

PubMed2000

Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes.

Bacteria producing endonuclease colicins are protected against their cytotoxic activity by virtue of a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. DNase binding by the immunity protein occurs through a "dual recognition" mechanism in which conserved residues from helix III act as the binding-site anchor, while variable residues from helix II define specificity. We now report the 1.7 A crystal structure of the 24.5 kDa complex formed between the endonuclease domain of colicin E9 and its cognate immunity protein Im9, which provides a molecular rationale for this mechanism. Conserved residues of Im9 form a binding-energy hotspot through a combination of backbone hydrogen bonds to the endonuclease, many via buried solvent molecules, and hydrophobic interactions at the core of the interface, while the specificity-determining residues interact with corresponding specificity side-chains on the enzyme. Comparison between the present structure and that reported recently for the colicin E7 endonuclease domain in complex with Im7 highlights how specificity is achieved by very different interactions in the two complexes, predominantly hydrophobic in nature in the E9-Im9 complex but charged in the E7-Im7 complex. A key feature of both complexes is the contact between a conserved tyrosine residue from the immunity proteins (Im9 Tyr54) with a specificity residue on the endonuclease directing it toward the specificity sites of the immunity protein. Remarkably, this tyrosine residue and its neighbour (Im9 Tyr55) are the pivots of a 19 degrees rigid-body rotation that relates the positions of Im7 and Im9 in the two complexes. This rotation does not affect conserved immunity protein interactions with the endonuclease but results in different regions of the specificity helix being presented to the enzyme.

Bacterial Proteins↗

PubMed2000

Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form.

Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S](2+/1+) cluster and one [3Fe-4S](1+/0) cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys(11)-Xaa-Xaa-Asp(14)-Xaa-Xaa-Cys(17)-Xaa(n)-Cys(51)-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S]<-->[4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg.l(-1)), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl(3) together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.

Amino Acid Sequence↗

PubMed2000

Slow formation of [3Fe-4S](1+) clusters in mutant forms of Desulfovibrio africanus ferredoxin III.

Desulfovibrio africanus ferredoxin III (Da FdIII) readily interconverts between a 7Fe and an 8Fe form with Asp-14 believed to provide a cluster ligand in the latter form. To investigate the factors important for cluster interconversion in Fe/S cluster-containing proteins we have studied two variants of Da FdIII produced by site-directed mutagenesis, Asp14Glu and Asp14His, with cluster incorporation performed in vitro. Characterisation of these proteins by UV/visible, EPR and (1)H NMR spectroscopies revealed that the formation of the stable 7Fe form of these proteins takes some time to occur. Evidence is presented which indicates the [4Fe-4S](2+) cluster is incorporated prior to the [3Fe-4S](1+) cluster.

Amino Acid Substitution↗

PubMed2000

The structure of TolB, an essential component of the tol-dependent translocation system, and its protein-protein interaction with the translocation domain of colicin E9.

BACKGROUND: E colicin proteins have three functional domains, each of which is implicated in one of the stages of killing Escherichia coli cells: receptor binding, translocation and cytotoxicity. The central (R) domain is responsible for receptor-binding activity whereas the N-terminal (T) domain mediates translocation, the process by which the C-terminal cytotoxic domain is transported from the receptor to the site of its cytotoxicity. The translocation of enzymatic E colicins like colicin E9 is dependent upon TolB but the details of the process are not known. RESULTS: We have demonstrated a protein-protein interaction between the T domain of colicin E9 and TolB, an essential component of the tol-dependent translocation system in E. coli, using the yeast two-hybrid system. The crystal structure of TolB, a procaryotic tryptophan-aspartate (WD) repeat protein, reveals an N-terminal alpha + beta domain based on a five-stranded mixed beta sheet and a C-terminal six-bladed beta-propeller domain. CONCLUSIONS: The results suggest that the TolB-box residues of the T domain of colicin E9 interact with the beta-propeller domain of TolB. The protein-protein interactions of other beta-propeller-containing proteins, the yeast yPrp4 protein and G proteins, are mediated by the loops or outer sheets of the propeller blades. The determination of the three-dimensional structure of the T domain-TolB complex and the isolation of mutations in TolB that abolish the interaction with the T domain will reveal fine details of the protein-protein interaction of TolB and the T domain of E colicins.

Amino Acid Sequence↗

PubMed2000

Characterisation of oxidised 7Fe dicluster ferredoxins with NMR spectroscopy.

Dicluster ferredoxins (Fds) from Sulfolobus acidocaldarius and Desulfovibrio africanus (FdIII) have been studied using 1H NMR. Both wild-type proteins contain a [3Fe-4S]+/0 and a [4Fe-4S]2+/+ cluster as isolated. The [4Fe-4S]2+/+ cluster (cluster II) is bound by cysteine residues arranged in a classic ferredoxin motif: CysI-(Xaa)2-CysII-(Xaa)2-CysIII-(Xaa)n-CysIV-Pro , whilst the binding motif of the [3Fe-4S]+/0 cluster (cluster I) has a non-ligating aspartic acid (Asp14) at position II, i.e. CysI-(Xaa)2-Asp-(Xaa)2-CysIII. D. africanus FdIII undergoes facile cluster transformation from the 7Fe form to the 8Fe form, but S. acidocaldarius Fd does not. Many factors determine the propensity of a cluster to undergo interconversion, including the presence, and correct orientation, of a suitable ligand. We have investigated this using 1H NMR by introducing a potential fourth ligand into the binding motif of cluster I of D. africanus FdIII. Asp14 has been mutated to cysteine (D14C), glutamic acid (D14E) and histidine (D14H). Cluster incorporation was performed in vitro. The cluster types present were identified from the chemical shift patterns and temperature-dependent behaviour of the hyperfine-shifted resonances. Factors influencing cluster ligation and cluster interconversion, in vitro, are discussed. Furthermore, the data have established that the residue at position II in the cluster binding motif of cluster I is influential in determining the chemical shift pattern observed for a [3Fe-4S]+ cluster when a short/symmetric binding motif is present. Based on this, a series of rules for characterising the 1H NMR chemical shifts of mono- and di-cluster [3Fe-4S]+ cluster-containing ferredoxins is given.

Amino Acid Motifs↗

PubMed2000

NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9.

The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions. We have used 1D 1H and 2D 1H-15N NMR methods to characterise the binding of Co2+, Ni2+ and Zn2+ to this protein. Data for the Co2+-substituted and Ni2+-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni2+-substituted protein show that two of the histidines are coordinated through their N(epsilon2) atoms and one via its N(delta1). Furthermore, the NMR spectrum of the Ni2+-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni2+, and by a change in pH, consistent with an ionisable group at the metal centre with a pKa of 7.9. Binding of an inhibitor protein to the DNase does not perturb the resonances of the metal site, suggesting there is no substantial conformation change of the DNase HNH motif on inhibitor binding. 1H-15N NMR data for the Zn2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different 1H-15N correlation NMR spectra, and that the binding of Zn2+ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region.

Binding Sites↗

PubMed2000

Rats exhibiting acute behavioural tolerance to nicotine have more [125I]alpha-bungarotoxin binding sites in brain than rats not exhibiting tolerance.

Adult male Sprague-Dawley rats trained to discriminate nicotine (0.4 mg/kg, s.c. vs vehicle) using a two-lever food-reinforced operant discriminative stimulus (DS) paradigm were tested as to the ability of each subject to develop acute tolerance to nicotine. Nicotine (0.8 mg/kg, s.c.) was administered to nicotine-trained rats in their home cage and each rat tested as to its ability to detect a 2nd dose of nicotine (0.4 mg/kg, s.c.) injected at 30 min intervals thereafter (90-180 min). Tolerance was determined by evaluating nicotine-correct responding during a 2 min test session. The results of this experiment indicated that 8 out of 31 rats (26%) displayed acute tolerance (desensitizers); 18 rats (58%) did not exhibit acute tolerance (non-desensitzers) and five rats (16%) fell into a middle group and were designated as neither desensitizers or non-desensitizers. The mode time for acute tolerance was 150 min, with each desensitizer rat displaying a unique temporal profile which was replicable 4-5 weeks later. Receptor autoradiographic analysis indicated no significant differences in [3H]epibatidine binding sites in the brains of desensitizers and non-desensitizers. In contrast, [125I]alpha-bungarotoxin binding was significantly higher in a number of brain regions in desensitizers. In situ hybridization analysis revealed no difference in alpha7 nAChR subunit mRNA levels between desensitizers and non-desensitizers. These observations can be interpreted to suggest that the ability to display acute tolerance to nicotine is contingent upon the ability to upregulate alpha7 nAChRs. These data may also be central to understanding the variability of tobacco use in humans, which may be contingent on the ability of the receptors binding to alpha-bungarotoxin to be responsive to nicotine-induced desensitization.

Animals↗

PubMed2000

Raltitrexed ('Tomudex') and radiotherapy can be combined as postoperative treatment for rectal cancer.

BACKGROUND: The optimal adjuvant therapy for operable rectal cancer is likely to be a combination of radiotherapy and chemotherapy. Raltitrexed ('Tomudex') is a specific thymidylate synthase inhibitor with a convenient administration schedule, acceptable and manageable toxicity, radiosensitising properties, and proven efficacy in the treatment of advanced colorectal cancer. It may, therefore, offer advantages compared with standard 5-FU chemotherapy regimens used in colorectal cancer. The aim of this phase I, dose-escalation study was to determine the recommended dose of raltitrexed for use with postoperative pelvic radiotherapy in patients with rectal cancer. PATIENTS AND METHODS: Patients with resected Dukes' stage B or C rectal cancer were treated with a combination of raltitrexed and radiotherapy (50.4 Gy at 1.8 Gy per fraction over five to six weeks). At least three patients were treated at each of three escalating raltitrexed dose levels (2.0, 2.6 and 3.0 mg/m2) once every three weeks. Toxicity was assessed by the recording of WHO adverse events and biochemistry and haematology determinations. RESULTS: A total of 22 patients entered the study, 17 of whom had Dukes' stage C disease. All three patients entered at a dose level of 3.0 mg/m2 experienced dose-limiting toxicity (DLT) (2 patients had grade 3 leucopenia and 1 patient had grade 2 leucopenia and grade 3 diarrhoea); however, only 2 of 1 patients entered at a dose level of 2.6 mg/m2 experienced DLT (1 patient had grade 4 neutropenia and 1 patient died probably due to aspiration pneumonia unrelated to treatment). The most common haematological toxic events were leucopenia (8 patients) and anaemia (6 patients). Only four haematological or biochemical toxic events were of grade 3 or 4. Other common toxicities were diarrhoea and nausea, which occurred in 15 and 9 patients, respectively. CONCLUSIONS: This study demonstrates that raltitrexed can be combined with postoperative radiotherapy for treatment of patients with Dukes' stage B or C rectal cancer. The recommended dose of raltitrexed in this setting is 2.6 mg/m2, which is close to the full monotherapy dose.

Adult↗

PubMed2000

Differentiating genomics companies.

As the value of raw sequence data diminishes, genomics companies are evolving new competencies as providers of information, therapeutic products, or novel technology.

Biotechnology↗

PubMed2000

A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells.

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.

Amino Acid Sequence↗

PubMed2000

Prevalence of malnutrition on admission to four hospitals in England. The Malnutrition Prevalence Group.

AIMS: The primary objective was to estimate prevalence of malnutrition on admission to four hospitals. Secondary objectives included assessing the relationship between nutritional status and length of hospital stay, numbers of new prescriptions, new infections and disease severity. METHODS: We entered eligible patients according to predefined quotas for elective and emergency admissions to 23 specialties. We measured height, weight, Body Mass Index and anthropometrics, and recorded history of unintentional weight loss. Patients who had lost > or = 10% of their body weight, had a Body Mass Index <20, or had a Body Mass Index <20 with one anthropometric measurement <15th centile were considered malnourished. RESULTS: Of 1611 eligible patients, 761 did not participate; 269 were too ill; 256 could not be weighed; and 236 refused consent. Eight hundred and fifty were subsequently evaluated. Prevalence of malnutrition on admission was 20%. Length of stay, new prescriptions and infections and disease severity were significantly higher in the malnourished. CONCLUSIONS: One patient in every five admitted to hospital is malnourished. Although this figure is unacceptably high, it may underestimate true prevalence. Malnutrition was associated with increased length of stay, new prescriptions and infections. Malnutrition may also have contributed to disease severity.

Adolescent↗

PubMed2000

Unusual fatal mechanisms in nonasphyxial autoerotic death.

The diagnosis of autoerotic death is most often made when there has been accidental asphyxia from ropes or ligatures used by the deceased as a part of his or her autoerotic ritual. Three cases of probable autoerotic death are reported in which the mechanisms of death involved hyperthermia, sepsis, and hemorrhage, respectively. Case 1: A 46-year-old man was found dead in bushland clothed in a dress, female undergarments, and seven pairs of stockings/pantyhose. The underwear had been cut to enable exposure of the genitals. The recorded daily maximum temperature was 39 degrees C, and the deceased had been taking the drug benztropine. Death was attributed to hyperthermia due to a combination of excessive clothing, high ambient temperature, and prescription drug side effect. Case 2: A 40-year-old man was found dead in his boarding house. At autopsy, a pencil was found within his abdominal cavity with perforation of the bladder and peritonitis. Death was attributed to peritonitis/sepsis following intraurethral introduction of a pencil. Case 3: A 56-year-old man was found dead lying on his bed following massive rectal hemorrhage. A blood stained shoe horn was found nearby. Death was attributed to hemorrhage following laceration of the anal canal with a shoe horn. The diagnosis of autoerotic death may be difficult when typical features are absent, however, any unusual injury associated with genitourinary manipulation must raise this possibility.

Adult↗

PubMed2000

Female gender associates with increased duration of intubation and length of stay after coronary artery surgery. CABG Clinical Benchmarking Database Participants.

BACKGROUND: Females have worse outcome than do males after coronary artery bypass grafting; however, gender effects on length of stay (LOS) outcomes, such as duration of intubation or intensive care unit (ICU) LOS, have not been evaluated previously. The authors hypothesized that adjustment for pertinent preoperative covariates would eliminate any significant effect of gender on duration of intubation, LOS in the ICU after extubation, total ICU LOS, postoperative (exclusive of ICU) LOS, or total postoperative LOS. METHODS: Patients undergoing elective or urgent primary coronary artery bypass grafting surgery at 51 academic health centers in 1995 and 1997 were studied. Unique multivariable statistical models were developed for duration of intubation, ICU LOS after extubation, total ICU LOS, and postoperative (exclusive of ICU and total) LOS to test for independent associations with gender. Preoperative but not intraoperative or postoperative variables were included in the model. P> or =0.01 was considered significant. RESULTS: All LOSs were of significantly longer duration in females than in males in both the 1995 (n = 1,064) and 1997 (n = 910) data collections. After covariate adjustment, female sex remained associated with significantly longer duration ICU LOS and total postoperative LOS in both the 1995 (female:male ratios 1.30:1 and 1.13:1, respectively) and the 1997 (female:male ratios 1.19:1 and 1.12:1, respectively) data sets. After covariate adjustment, duration of intubation and ICU LOS after extubation were of significantly longer duration in women than men in 1995 (female:male ratios 1.22:1 and 1.39:1, respectively), but the differences were not significant in 1997. CONCLUSIONS: Even in the context of accelerated recovery programs, these analyses show that female sex has powerful associations with increased LOS intervals for coronary artery bypass grafting surgery, even after adjustment for preoperative covariates. These effects could result from differences in the ways in which men and women respond to coronary artery disease, anesthesia, and coronary artery bypass grafting surgery, or to bias on the part of healthcare workers.

Aged↗

PubMed2000

Crystallization of the cytotoxic domain of a ribosome-inactivating colicin in complex with its immunity protein.

The complex between the ribonuclease domain of the ribosome-inactivating colicin E3 and its protein inhibitor, the cognate immunity Im3, has been crystallized and preliminary X-ray characterization has been performed. Single crystals of the 1:1 complex were grown from hanging-drop vapour-diffusion experiments using 2-propanol as a precipitant. The space group is P3(1)21 or P3(2)21, with unit-cell parameters a = b = 93.7, c = 76.2 A. When cryocooled, these crystals diffract to a resolution of 2.4 A. A search for suitable conventional heavy-atom derivatives was unsuccessful and so Im3 mutants containing engineered cysteine or methionine residues have been produced for mercury soaks and selenomethionine-labelling experiments, respectively.

Amino Acid Substitution↗

PubMed2000

Slow conformational dynamics of an endonuclease persist in its complex with its natural protein inhibitor.

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of chemical exchange cross peaks in 750 MHz 15N-1H-15N HSQC-NOESY-HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange. The rate of exchange from the major to the minor form was determined to be 1.1 +/- 0.2 s(-1) at 298 K. Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 +/- 0.11 s(-1) at 288 K, and that the bound Im9 is fixed in one conformation. The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 A radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange. The possible functional significance of this feature of the DNase is discussed.

Crystallography, X-Ray↗

PubMed2000

NMR investigation of the interaction of the inhibitor protein Im9 with its partner DNase.

The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3-84 were 0.76 +/- 0.14 A for the backbone atoms and 1.36 +/- 0.15 A for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 A, respectively, for residues 4-83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H-1H-13C NOESY-HSQC spectra with and without 13C decoupling. The NMR-defined interface agrees well with that determined in a recent X-ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.

Amino Acid Sequence↗