Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Geobacillus”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources
PubMed1996

Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA: II. Specificity determinants of RNA-protein recognition.

S15 is a primary ribosomal protein that interacts specifically with a three-way junction in the central domain of 16 S rRNA, whose binding induces a conformational change in the RNA. In the accompanying paper, we demonstrated that S15 binds with high affinity to a 61 nucleotide RNA corresponding to the minimal rRNA binding site. Here, the sequence and structural determinants for the RNA in the Bacillus stearothermophilus S15-rRNA interaction have been probed using site-directed mutagenesis, chemical modification interference, and iodine footprinting of phosphorothioate RNA. Mutations and RNA modifications that interfere with protein binding cluster in two distinct regions, one containing an internal loop and the other containing a three-way junction. The internal loop, defined by two A.G base-pairs and a bulged guanosine, is not important for the specific interaction, however, BS15 interacts with a phylogenetically conserved G.U base-pair above this internal loop. Near the three-way junction in helix 22, a bulged adenosine and two base-pairs adjacent to the junction also provide important determinants for BS15 binding. Chemical modification interference also suggests that four highly phylogenetically conserved nucleotides in the three-way junction may form non-canonical G.G and U.A base-pairs that are required for the BS15-rRNA interaction. Ethylation modification interference suggests that BS15 binding is accompanied by a conformational change in the RNA involving orientation of helices 20 and 22 at an acute angle with respect to one another. Projection of the data provided by mutagenesis, chemical modification interference analysis, and iodine footprinting onto a three-dimensional model illustrates that BS15 is likely to interact with the minor groove along an extended face of helix 22.

Base Sequence↗
PubMed1996

Recognition of a surface loop of the lipoyl domain underlies substrate channelling in the pyruvate dehydrogenase multienzyme complex.

In the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus, the interaction between the pyruvate decarboxylase (E1p) component and the lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component was investigated using a combination of site-directed mutagenesis and NMR spectroscopy. Residues 11 to 15 (EGIHE) of the lipoyl domain, part of a surface loop close in space to the beta-turn containing the lipoyl-lysine residue (position 42), were deleted or replaced. The mutant domains all retained their three-dimensional structures and ability to become lipoylated, but in the absence of the loop the lipoyl-lysine residue could no longer be reductively acetylated by E1p. A mutation (N40A) in the N- terminal part of the lipoyl-lysine hairpin showed that it is involved in recognition of the domain by E1p but other mutations in the loop (E15A) and close to the lipoyl-lysine hairpin (V44S, V45S and E46A) were without effect. The heteronuclear multiple quantum coherence NMR spectra of 15N-labelled lipoyl domain in the presence and absence of B. stearothermophilus E1p were recorded. Of the 85 amino acid residues in the lipoyl domain, 13 exhibited significant differences in chemical shift. These differences, most of which were associated with residues in the surface loop between positions 8 and 15 and in, or close to, the lipoyl-lysine hairpin, indicate that E1p makes contact with the lipoyl domain in these areas. The combined results of directed mutagenesis and NMR spectroscopy point to the surface loop as a major determinant of the interaction of lipoyl domain with E1p. The specificity of this essential interaction provides the molecular basis of substrate channelling in this, the first committed, step of the enzyme reaction mechanism.

Acetylation↗
PubMed1996

Guided evolution of enzymes with new substrate specificities.

A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alpha E-beta D) of Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH). All but one of 38 enzyme variants examined were thermally stable and had native-like hydrodynamic properties. In this sample, there was no bias detected in either the DNA or amino acid sequences encoded. We argue that the alpha E-beta D surface loop sequence is unimportant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities. The selection of NAD-dependent dehydrogenases with specificity for: malate, phenyllactate, hydroxyisocaproate and 4-phenyl-2-hydroxy-butanoate from two bsLDH libraries is described. This required a highly discriminatory screen for 2-hydroxy acid dehydrogenase activity to select enzymes which, in the absence of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition. In general the amino acid residues at positions 101 and 102 which determined substrate specificity were as expected from hydrophobic and ionic complementarity to the substrate. For example, a bsLDH variant with Asn101Va1102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate. Using molecular modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate. Similarly, nine out of ten malate dehydrogenases (MDHs) selected had an arginine residue at position 102 to complement the negatively charged carboxyl group in oxaloacetate. One, Arg101Arg102 (Kcat/Kmoxaloacetate = 1.6 x 10(6) M-1 S-1) is 25% more active than the previous best synthetic MDH. There were surprises: present understanding would not have predicted the oxaloacetate transforming activity of Ser101Leu102 or the phenylpyruvate activity of Pro101Lys102. The former is about one-third as efficient as the best malate dehydrogenase selected, whilst the latter had about one-seventh of the best phenylpyruvate dehydrogenase activity.

Amino Acid Sequence↗
PubMed1996

Ribosomal protein L9: a structure determination by the combined use of X-ray crystallography and NMR spectroscopy.

The structure of protein L9 from the Bacillus stearothernophilus ribosome has been determined at 2.5 A resolution by refinement against single crystal X-ray diffraction data with additional constraints provided by NMR data. This highly elongated protein consists of two domains separated by a nine-turn connecting helix. Conserved aromatic and positively charged amino acid residues on the surface of each domain are likely to be directly involved in binding 23 S ribosomal RNA. The shape of the protein, with its two widely spaced RNA-binding sites, suggests that it may serve as a "molecular strut", most likely playing a role in ribosome assembly and/or maintaining the catalytically active conformation of the ribosomal RNA. The combined use of X-ray and NMR data in the refinement procedure was essential in defining the N-terminal domain of the protein, which was relatively poorly determined by the X-ray data alone. In addition to resolving the ambiguities in defining the hydrophobic core and side-chain conformations with the N-terminal domain, this combined NMR-X-ray analysis provides the first detailed and accurate view of the N-terminal RNA-binding site. NMR data also showed that the N-terminal domain is stable in solution, indicated by amide protons that are protected from solvent exchange. The lack of definition of the N-terminal domain in the X-ray structure is therefore likely due to packing disorder within the crystal rather than structural instability. This combined NMR-X-ray analysis provides a useful model as to how X-ray and NMR data can be practically and logically combined in the determination of the structure of a single protein molecule.

Amino Acid Sequence↗
PubMed1997

Structure-function relationships within the peptide deformylase family. Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion.

Thermus thermophilus peptide deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure.

Amidohydrolases↗
PubMed1997

The stability and dynamics of ribosomal protein L9: investigations of a molecular strut by amide proton exchange and circular dichroism.

Nuclear magnetic resonance and circular dichroism experiments were used to investigate the stability and dynamic aspects of ribosomal protein L9 from Bacillus stearothermophilus in solution. This unusually shaped protein, with its two widely spaced RNA-binding domains linked by a connecting helix, has been hypothesized to serve as a "molecular strut", most likely playing a role in ribosome assembly and/or maintaining the catalytically active conformation of ribosomal RNA. Protection factors for amide proton exchange were quantitatively measured in an extensive series of NMR experiments, providing probes of the stability and dynamics of localized regions of the protein. Results show that each of the two RNA-binding domains contains a highly stable core. The exposed central helix that connects the two domains is helical in solution, albeit not rigid, a result that is supported by amide proton protection factors, circular dichroism measurements, and carbon-13 and proton chemical shift index values. A conserved glycine and lysine-rich loop in the N-terminal domain is ordered and quite stable, a surprising result, since this loop had been presumed to be disordered in the original crystallographic analysis. Interestingly, the most dynamic parts of the protein are the regions that contain the likely RNA-binding residues in each of the two domains. The present results add further support to the notion that the L9 protein plays an architectural role within the ribosome, with the central helix serving as a molecular strut, or perhaps a spring, linking the two widely spaced RNA-binding domains.

Amides↗
PubMed1997

A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+.

Mutations have been introduced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order to convert its cofactor selectivity from a specificity towards NAD into a preference for NADP. In the B-S mutant, five mutations (L33T, T34G, D35G, L187A, P188S) were selected on the basis of a sequence alignment with NADP-dependent chloroplastic GAPDHs. In the D32G-S mutant, two of the five mutations mentioned above (L187A, P188S) have been used in combination with another one designed from electrostatic considerations (D32G). Both mutants exhibit a dual-cofactor selectivity at the advantage of either NAD (B-S) or NADP (D32G-S). In order to analyse the cofactor-binding site plasticity at the molecular level, crystal structures of these mutants have been solved, when complexed with either NAD+ (D32G-Sn, resolution 2.5 A, R = 13.9%; B-Sn, 2.45 A, 19.3%) or NADP+ (D32G-Sp, 2.2 A, 19.2%; B-Sp, 2.5 A, 14.4%). The four refined models are very similar to that of the wild-type GAPDH and as expected resemble more closely the holo form than the apo form. In the B-S mutant, the wild-type low affinity for NADP+ seems to be essentially retained because of repulsive electrostatic contacts between the extra 2'-phosphate and the unchanged carboxylate group of residue D32. Such an antideterminant effect is not well compensated by putative attractive interactions which had been expected to arise from the newly-introduced side-chains. In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme. Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH. In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type. The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group. As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188. When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP. In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.

Binding Sites↗
PubMed1997

Species-specific tRNA recognition in relation to tRNA synthetase contact residues.

In spite of variations in the sequences of tRNAs, the genetic code (anticodon trinucleotides) is conserved in evolution. However, non-anticodon nucleotides which are species specific are known to prevent a given tRNA from functioning in all organisms. Conversely, species-specific tRNA contact residues in synthetases should also prevent cross-species acylation in a predictable way. To address this question, we investigated the relatively small tyrosine tRNA synthetase where contacts of Escherichia coli tRNA(Tyr) with the alpha2 dimeric protein have been localized by others to four specific sequence clusters on the three-dimensional structure of the Bacillus stearothermophilus enzyme. We used specific functional tests with a previously not-sequenced and not-characterized Mycobacterium tuberculosis enzyme and showed that it demonstrates species-specific aminoacylation in vivo and in vitro. The specificity observed fits exactly with the presence of the clusters characteristic of those established as important for recognition of E. coli tRNA. Conversely, we noted that a recent analysis of the tyrosine enzyme from the eukaryote pathogen Pneumocystis carinii showed just the opposite species specificity of tRNA recognition. According to our alignments, the sequences of the clusters diverge substantially from those seen with the M. tuberculosis, B. stearothermophilus and other enzymes. Thus, the presence or absence of species-specific residues in tRNA synthetases correlates in both directions with cross-species aminoacylation phenotypes, without reference to the associated tRNA sequences. We suggest that this kind of analysis can identify those synthetase-tRNA covariations which are needed to preserve the genetic code. These co-variations might be exploited to develop novel antibiotics against pathogens such as M. tuberculosis and P. carinii.

Acylation↗
PubMed1997

An exceptionally stable helix from the ribosomal protein L9: implications for protein folding and stability.

The ribosomal protein L9 has an unusual structure comprising two compact globular domains connected by a 34 residue alpha-helix. The middle 17 residues of the helix are exposed to solvent while the first seven pack against and form part of the N-terminal domain, and the last ten form part of the C-terminal domain. Here we report results which show that a peptide corresponding to the central helix of L9 is monomeric in aqueous solution and >85% helical at 1 degrees C and 68(+/-7)% helical at 25 degrees C. This is considerably more helical than any other protein fragment studied to date. Another peptide corresponding to the middle 17 residues of the helix is monomeric and is 41(+/-4)% helical at 1 degrees C. Because the central helix has high intrinsic stability the globular N and C-terminal domains will likely be stabilized by their interactions with the helix. Therefore, the stability of the two terminal domains should not be completely independent because both domains gain stability from a shared structural element, the central helix. Also, the ability of the central helix to form native-like structure in isolation highlights a potential role for the helix in the early stages of the folding process.

Amino Acid Sequence↗
PubMed1998

Protein and Mg(2+)-induced conformational changes in the S15 binding site of 16 S ribosomal RNA.

The Bacillus stearothermophilus ribosomal protein S15 binds to the central domain of the 16 S rRNA inducing a conformational change in a three-way helical junction. To understand the nature of this conformational change, extended-helical junctions were prepared to examine the effects of S15 or Mg2+ binding on the relative helical orientation using native gel electrophoretic mobility and transient electric birefringence. The free junction is planar with approximately 120 degrees interhelical angles, whereas S15 and Mg2+ yield a junction conformation that remains planar in which two helices, 21 and 22, become colinear and the third, helix 20, forms a 60 degrees angle with respect to helix 22. This conformational change is thought to be important for directing the assembly of the central domain of the 30 S ribosomal subunit.

Base Sequence↗
PubMed1998

Cooperativity in Bacillus stearothermophilus pyruvate kinase.

The enzyme pyruvate kinase (PK) from the moderate thermophile Bacillus stearothermophilus has been used as a model system with which to investigate the homotropic and heterotropic cooperative interactions of the enzyme. Cooperative ligand binding by the wild-type enzyme was measured using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative structural changes induced by the substrate phosphoenolpyruvate (PEP) are distinct from those induced by the allosteric activator ribose- 5-phosphate (R5P). Furthermore the structural transition induced by the binding of saturating amounts of both PEP and R5P is itself distinct. This conclusion was further substantiated by the production of five mutant proteins in which the R5P- and PEP-induced homotropic cooperative transitions were separated. These results suggest that the cooperativity exhibited by pyruvate kinase from B. stearothermophilus does not conform to a simple two-state model. A putative four-state model is proposed.

Amino Acid Sequence↗
PubMed1998

On the global architecture of initiation factor IF3: a comparative study of the linker regions from the Escherichia coli protein and the Bacillus stearothermophilus protein.

Initiation factor IF3 is a protein involved in the initiation stage of protein synthesis. It consists of two global domains linked by a 20 residue long, solvent-exposed linker. Recently, the structure of the N and C-terminal domains of the Bacillus stearothermophilus protein have been solved by X-ray crystallography and the structure of the intact Escherichia coli protein has been studied by NMR. These two studies have led to apparently contradictory models for the domain organization of IF3. The NMR study of the E. coli protein indicates that the linker region is flexible, while the studies of the isolated N and C-terminal domains of the B. stearothermophilus protein suggest that the linker forms a rigid helical rod. In order to resolve this discrepancy, a set of peptides corresponding to the linker regions of the B. stearothermophilus and the E. coli protein were synthesized. Circular dichroism and NMR spectroscopy were used to study the helical content as a function of pH, temperature, peptide concentration and ionic strength. Both peptides are monomeric. The estimated helical content of the linker fragment from B. stearothermophilus is 68% at high pH and 1 degree C. The measured helicity decreases to 53% at pH 7.0 and 1 degree C. In contrast, the peptide corresponding to the E. coli IF3 linker region is largely unstructured with a maximum helical content of 15% at high pH and only 8% at pH 7.0, 1 degree C. These results suggest that the different structures observed for the two intact proteins may be due to the different intrinsic stability of the two linker peptides. The helical content of the two linker peptides is, however, much closer when the peptides are compared at the respective temperatures of optimum growth for E. coli and B. stearothermophilus (3% versus 17%). The pH and ionic strength dependence of the helical content of the B. stearothermophilus peptide demonstrates that side-chain/side-chain interactions play an important role in stabilizing the helical structure. In addition, studies with mutant peptides show that the first Asp residue in the linker sequence helps to stabilize the helix via an N- capping interaction.

Amino Acid Sequence↗
PubMed1998

Crystal structure of ribosomal protein S8 from Thermus thermophilus reveals a high degree of structural conservation of a specific RNA binding site.

S8 is one of the core ribosomal proteins. It binds to 16 S RNA with high affinity and independently of other ribosomal proteins. It also acts as a translational repressor in Escherichia coli by binding to its own mRNA. The structure of Thermus thermophilus S8 has been determined by the method of multiple isomorphous replacement at 2.9 A resolution and refined to a crystallographic R-factor of 16.2% (Rfree 27.5%). The two domains of the structure have an alpha/beta fold and are connected by a long protruding loop. The two molecules in the asymmetric unit of the crystal interact through an extensive hydrophobic core and form a tightly associated dimer, while symmetry-related molecules form a joint beta-sheet of mixed type. This type of protein-protein interaction could be realized within the ribosomal assembly. A comparison of the structures of T. thermophilus and Bacillus stearothermophilus S8 shows that the interdomain loop is eight residues longer in the former and reveals high structural conservation of an extensive region, located in the C-terminal domain. From mutational studies this region was proposed earlier to be involved in specific interaction with RNA. On the basis of these data and on the comparison of the two structures of S8, it is proposed that the three-dimensional structure of specific RNA binding sites in ribosomal proteins is highly conserved among different species.

Amino Acid Sequence↗
PubMed1998

An investigation of the dynamics of ribosomal protein L9 using heteronuclear NMR relaxation measurements.

The dynamic properties of ribosomal protein L9 from Bacillus stearothermophilus were investigated in solution using an analysis of nitrogen-15 longitudinal and transverse relaxation rates and amide nitrogen-proton nuclear Overhauser effects. The relaxation rates of the amide nitrogen nuclei were found to be correlated with the angle between the amide nitrogen-proton bond vectors and the long axis of the protein. This directional dependence of the nuclear relaxation rates is consistent with the protein having a highly elongated shape in solution, consistent with that observed in previous X-ray crystallographic studies of the crystalline form. Analysis of the nuclear relaxation data shows that the solvent-exposed nine-turn alpha helix connecting the two domains has a relatively high degree of order, in contrast to the connecting helix in the similarly shaped, but functionally different, calmodulin protein. The rotational correlation times associated with the amide nitrogen atoms of the N-terminal domain are on average slightly shorter than those of the C-terminal domain and connecting helix, providing evidence that the N-terminal domain exhibits some degree of independence in tumbling, in addition to other fast internal motions. The putative RNA-binding surfaces in each of the protein domains are characterized by relatively low order parameters, indicating that these are the most flexible regions of the molecule. Overall, the picture of the internal dynamics provided by nuclear relaxation measurements is similar to that obtained from a detailed study of amide proton exchange rates, but differs markedly from the picture provided by crystallographic temperature factors. The present study describes a molecule with unusual and complex dynamic properties, and supports a model where the protein functions as a "molecular strut" within the ribosome.

Amino Acid Sequence↗
PubMed1998

Structure and expression of elongation factor Tu from Bacillus stearothermophilus.

The tuf gene coding for elongation factor Tu (EF-Tu) of Bacillus stearothermophilus was cloned and sequenced. This gene maps in the same context as the tufA gene of Escherichia coli str operon. Northern-blot analysis and primer extension experiments revealed that the transcription of the tuf gene is driven from two promoter regions. One of these is responsible for producing a 4.9-kb transcript containing all the genes of B. stearothermophilus str operon and the other, identified adjacent to the stop codon of the fus gene and designated tufp, for producing a 1.3-kb transcript of the tuf gene only. In contrast to the situation in E. coli, the ratio between the transcription products was found to be about 10:1 in favour of the tuf gene transcript. This high transcription activity from the tufp promoter might be accounted for by the presence of an extremely A+T-rich block consisting of 29 nucleotides which immediately precedes the consensus -35 region of the promoter. A very similar tuf gene transcription strategy and the same tufp promoter organization with the identical A/T block were found in Bacillus subtilis. The tuf gene specifies a protein of 395 amino acid residues with a molecular mass of 43,290 Da, including the N-terminal methionine. A computer-generated three-dimensional homology model shows that all the structural elements essential for binding guanine nucleotides and aminoacyl-tRNA are conserved. The presence of serine at position 376 and a low affinity for kirromycin determined by zone-interference gel electrophoresis (Kd approximately 8 microM) and by polyacrylamide gel electrophoresis under non-denaturing conditions are in agreement with the reported resistance of this EF-Tu to the antibiotic. The replacement of the highly conserved Leu211 by Met was identified as a possible cause of pulvomycin resistance.

Amino Acid Sequence↗
PubMed1998

Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9.

The folding and unfolding kinetics of the N-terminal domain of the ribosomal protein L9 have been measured at temperatures between 7 and 85 degrees C and between 0 and 6 M guanidine deuterium chloride. Stopped-flow fluorescence was used to measure rates below 55 degrees C and NMR lineshape analysis was used above 55 degrees C. The amplitudes and rate profiles of the stopped-flow fluorescence experiments are consistent with a two-state folding mechanism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneously to the simple model k=D exp(-DeltaG*/RT) where DeltaG* represents the change in apparent free energy between the transition state and the folded or unfolded state and D represents the maximum possible folding speed. DeltaG* is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to model stability changes with temperature. Approximately 60% of the surface area buried upon folding is buried in the transition state as evidenced by changes in the heat capacity and m value between the unfolded state and the transition state. The equilibrium thermodynamic parameters, DeltaCp degrees, m and DeltaG degrees, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-state. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius temperature dependence typical of globular proteins. When the folding rates are examined along constant DeltaG degrees/T contours they display an Arrhenius temperature dependence with a slope of -8600 K. This indicates that for this system, the non-Arrhenius temperature dependence of folding can be accounted for by the anomalous temperature dependence of the interactions which stabilize proteins.

Bacterial Proteins↗
PubMed1998

Characterisation of low free-energy excited states of folded proteins.

It is demonstrated that the identity of residues accessing excited conformational states that are of low free energy relative to the ground state in proteins can be obtained from amide proton NMR chemical shift temperature dependences displaying significant curvature. For the N-terminal domain of phosphoglycerate kinase, hen egg-white lysozyme and BPTI, conformational heterogeneity arises from a number of independent sources, including: structural instability resulting from deletion of part of the protein; a minor conformer generated through disulphide bond isomerisation; an alternative hydrogen bond network associated with buried water molecules; alternative hydrogen bonds involving backbone amides and surface-exposed side-chain hydrogen bond acceptors; and the disruption of loops, ends of secondary structural elements and chain termini. In many of these cases, the conformational heterogeneity at these sites has previously been identified by X-ray and/or NMR studies, but conformational heterogeneity of buried water molecules has hitherto received little attention. These multiple independent low free-energy excited states each involve a small number of residues and are shown to be within 2.5 kcal mol-1 of the ground state. Their relationship with the partially unfolded forms previously characterised using amide proton exchange studies is discussed.

Amides↗
PubMed1999

Functional domains of an NAD+-dependent DNA ligase.

Limited proteolysis of the NAD+-dependent DNA ligase from Bacillus stearothermophilus with thermolysin results in two fragments which were resistant to further proteolysis. These fragments were characterised by N-terminal protein sequencing and electrospray mass spectrometry. The larger, N-terminal fragment consists of the first 318 residues and the smaller, C-terminal fragment begins at residue 397 and runs to the C terminus. Both fragments were over-expressed in Escherichia coli and purified to homogeneity from this source. The large fragment retains the full self-adenylation activity of the intact enzyme, has minimal DNA binding activity and vastly reduced ligation activity. The small fragment lacks adenylation activity but binds to nicked DNA with a similar affinity to that of the intact enzyme. It is unable to stimulate the ligation activity of the large fragment. Atomic absorption spectroscopy showed that the intact protein and the small fragment bind a zinc ion but the large fragment does not. No evidence of any interaction between the two fragments could be obtained. Thus, we conclude that NAD+-dependent DNA ligases consist of at least two discrete functional domains: an N-terminal domain which is responsible for cofactor binding and self adenylation, and a C-terminal DNA-binding domain which contains a zinc binding site.

Adenine↗