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Biomedical subjects

S E Jackson

Publications and source records attributed to S E Jackson.

At least 19 recordsLinked to original sources
PubMed2000

Non-chondritic distribution of the highly siderophile elements in mantle sulphides.

The abundances of highly siderophile (iron-loving) elements (HSEs) in the Earth's mantle provide important constraints on models of the Earth's early evolution. It has long been assumed that the relative abundances of HSEs should reflect the composition of chondritic meteorites--which are thought to represent the primordial material from which the Earth was formed. But the non-chondritic abundance ratios recently found in several types of rock derived from the Earth's mantle have been difficult to reconcile with standard models of the Earth's accretion, and have been interpreted as having arisen from the addition to the primitive mantle of either non-chondritic extraterrestrial material or differentiated material from the Earth's core. Here we report in situ laser-ablation analyses of sulphides in mantle-derived rocks which show that these sulphides do not have chondritic HSE patterns, but that different generations of sulphide within single samples show extreme variability in the relative abundances of HSEs. Sulphides enclosed in silicate phases have high osmium and iridium abundances but low Pd/Ir ratios, whereas pentlandite-dominated interstitial sulphides show low osmium and iridium abundances and high Pd/Ir ratios. We interpret the silicate-enclosed sulphides as the residues of melting processes and interstitial sulphides as the crystallization products of sulphide-bearing (metasomatic) fluids. We suggest that non-chondritic HSE patterns directly reflect processes occurring in the upper mantle--that is, melting and sulphide addition via metasomatism--and are not evidence for the addition of core material or of 'exotic' meteoritic components.

Journal Article↗
PubMed1999

Folding pathway of FKBP12 and characterisation of the transition state.

The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12, with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (kF=4 s(-1)at 25 degrees C). The topological crossing of two loops in FKBP12, a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway. The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. In addition, unfolding and refolding studies in the presence of ligand provided information on the structure of the ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70 % of the surface area buried. The ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the alpha-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper).

Binding Sites↗
PubMed1999

Mapping the interactions present in the transition state for unfolding/folding of FKBP12.

The structure of the transition state for folding/unfolding of the immunophilin FKBP12 has been characterised using a combination of protein engineering techniques, unfolding kinetics, and molecular dynamics simulations. A total of 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. The transition state for folding is compact compared with the unfolded state, with an approximately 30 % increase in the native solvent-accessible surface area. All of the interactions are substantially weaker in the transition state, as probed by both experiment and molecular dynamics simulations. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state; instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domain from alpha-spectrin. For FKBP12, the central three strands of the beta-sheet, beta-strand 2, beta-strand 4 and beta-strand 5, comprise the most structured region of the transition state. In particular Val101, which is one of the most highly buried residues and located in the middle of the central beta-strand, makes approximately 60 % of its native interactions. The outer beta-strands and the ends of the central beta-strands are formed to a lesser degree. The short alpha-helix is largely unstructured in the transition state, as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side-chains within beta-strands 2, 4 and 5, and the C terminus of the alpha-helix. The precise residues involved in the nucleus differ in the two simulated transition state ensembles, but the interacting regions of the protein are conserved. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. The two independently derived transition state ensembles are structurally similar, which is consistent with a Bronsted analysis confirming that the transition state is an ensemble of states close in structure.

Computer Simulation↗
PubMed1999

Elemental composition of human milk from mothers of premature and full-term infants during the first 3 months of lactation.

To examine longitudinal and gestational effects of mineral content in human milk, we analyzed human milk from lactating mothers of premature (PRT, n = 24, < 2000 g birth weight, < 37 wk gestation) and full-term (FT, n = 19, > 2500 g, 39-41 wk gestation), living in Newfoundland, Canada. Samples were collected once a week for 8 wk with one final sample collected at 3 mo. Milk samples collected in acid-washed containers were wet ashed with concentrated HNO3, and barium, cadmium, calcium, cesium, cobalt, copper, cerium, lanthanum, magnesium, manganese, molybdenum, nickel, lead, rubidium, tin, strontium, and zinc were measured using inductively coupled plasma-mass spectrometry. Data were analyzed using standard multiple-regression procedures with correlated data analyses to take account of the relationship between successive weeks. Results indicated lower Ca and Pb in PRT milk. Calcium was the only nutritionally significant element to differ between groups. Molybdenum in both PRT and FT milk showed a definite decrease with time, suggesting that the Mo content in milk is homeostatically regulated. However, Ce, La, Ba, and Sn did not display any pattern indicative of biological regulation and potential human requirement.

Female↗
PubMed1999

Does trifluoroethanol affect folding pathways and can it be used as a probe of structure in transition states?

Nonaqueous co-solvents, particularly 2,2,2-trifluoroethanol (TFE), have been used as tools to study protein folding. By analyzing FKBP12, an alpha/beta-protein that folds with two-state kinetics, we have been able to address three key questions concerning the use of TFE. First, does TFE perturb the folding pathway? Second, can the observed changes in the rate of folding and unfolding in TFE be attributed to a change in free energy of a single state? Finally, can TFE be used to infer information on secondary structure formation in the transition state? Protein engineering experiments on FKBP12, coupled with folding and unfolding experiments in 0% and 9.6% TFE, conclusively show that TFE does not perturb the folding pathway of this protein. Our results also suggest that the changes in folding and unfolding rates observed in 9.6% TFE are due to a global effect of TFE on the protein, rather than the stabilization of any elements of secondary structure in the transition state. Thus, studies with TFE and other co-solvents can be accurately interpreted only when combined with other techniques.

Immunophilins↗
PubMed1998

Context-dependent nature of destabilizing mutations on the stability of FKBP12.

The context-dependent nature in which mutations affect protein stability was investigated using the FK506-binding protein, FKBP12. Thirty-four mutations were made at sites throughout the protein, including residues located in the hydrophobic core, the beta-sheet, and the solvent-exposed face of the alpha-helix. Urea-induced denaturation experiments were used to measure the change in stability of the mutants relative to that of the wild type (Delta DeltaGU-F). The results clearly show that the extent of destabilization, or stabilization, is highly context-dependent. Correlations were sought in order to link Delta DeltaGU-F to various structural parameters. The strongest correlation found was between Delta DeltaGU-F and N, the number of methyl(ene) groups within a 6 A radius of the group(s) deleted. For mutations of buried hydrophobic residues, a correlation coefficient of 0.73 (n = 16,where n is the number of points) was obtained. This increased to 0.81 (n = 24) on inclusion of mutations of partially buried hydrophobic residues. These data could be superimposed on data obtained for other proteins for which similarly detailed studies have been performed. Thus, the contribution to stability from hydrophobic side chains, independent of the extent to which a side chain is buried, can be estimated quantitatively using N. This correlation appears to be a general feature of all globular proteins. The effect on stability of mutating polar and charged residues in the alpha-helix and beta-sheet was also found to be highly context-dependent. Previous experimental and statistical studies have shown that specific side chains can stabilize the N-caps of alpha-helices in proteins. Substitutions of Ile56 to Thr and Asp at the N-cap of the alpha-helix of FKBP12, however, were found to be highly destabilizing. Thus, the intrinsic propensities of an amino acid for a particular element of secondary structure can easily be outweighed by tertiary packing factors. This study highlights the importance of packing density in determining the contribution of a residue to protein stability. This is the most important factor that should be taken into consideration in protein design.

Arginine↗
PubMed1998

How do small single-domain proteins fold?

Many small, monomeric proteins fold with simple two-state kinetics and show wide variation in folding rates, from microseconds to seconds. Thus, stable intermediates are not a prerequisite for the fast, efficient folding of proteins and may in fact be kinetic traps and slow the folding process. Using recent studies, can we begin to search for trends which may lead to a better understanding of the protein folding process?

Dimerization↗
PubMed1998

Health outcomes variables important to patients in the treatment of erectile dysfunction.

PURPOSE: Erectile dysfunction is underreported and the rate of noncompliance with therapy is high. The National Institutes of Health Consensus Conference on Impotence endorsed the need for outcomes research of the various approaches to treatment. The purpose of our exploratory study was to begin that process through identification of erectile dysfunction treatment outcomes variables that are important to men. MATERIALS AND METHODS: The study format consisted of focus group meetings. The goal of the discussions was to identify variables that are important to men when choosing among and judging the success of treatment modalities for erectile dysfunction. After discussion participants were individually asked to identify the 10 variables that they considered important and rank the 5 most important variables. An importance score reflecting group consensus was calculated for each variable. RESULTS: Success and negative outcomes were identified as the 2 most important outcomes variables for all treatment modalities. Participants defined success in several ways. The most important measures of success were cure, pleasure and partner satisfaction. Negative outcome was defined as negative consequences associated with treatment. CONCLUSIONS: The focus group discussion assisted in identifying and defining outcomes variables important to patients with erectile dysfunction. We believe that understanding issues and outcomes important to such patients is necessary to increase compliance with treatment and, thereby, increase the success of treatment of this widespread condition.

Adult↗
PubMed1996

Laboratory gloves as a source of trace element contamination.

Contamination in a trace element laboratory can come from a variety of sources, including laboratory gloves. Therefore, vinyl and latex gloves were obtained from as many manufacturers as would supply gloves. These gloves were either prepared for acid-washing and subsequent soaking in an acid solution, or immersed in an acid solution for a duration of either 1 min or 1 h. Incubation washes were analyzed for a variety of trace elements by flame atomic absorption spectroscopy (AAS) or inductively coupled mass spectrometry (ICP-MS). Results indicated that only three brands of vinyl gloves were acceptable for use in a trace element laboratory, whereas others had contamination of different elements. Latex gloves contained such high levels of biologically important elements that they were not considered suitable for routine trace element work. Vinyl gloves of choice should be routinely acid-washed before use in a trace element laboratory.

Air Pollution, Indoor↗
PubMed1996

Elemental fractionation in laser ablation inductively coupled plasma mass spectrometry.

The major challenge to the use of laser ablation sample introduction, combined with inductively coupled plasma mass spectrometry, is the problem of calibration. In the geological analysis of minerals, calibration is complicated by the extraordinarily wide variety of sample matrices which may be encountered. While there is a lack of mineral standards with well characterized concentrations near 1 microg/g, the NIST glass reference materials (SRM 610-617) have been demonstrated to be very useful for the analysis of a wide variety of lithophile elements in silicate samples. An internal reference element, for which the concentration is known in the sample, has been widely used to make corrections for the multiplicative effects of volume (or weight) of the sample ablated, instrument drift, and matrix effects. This procedure works extremely well where elements being determined and the internal reference element being used share similar ablation behaviours; i.e., they do not fractionate progressively during the ablation and transport process. In this study, it is demonstrated that, in terms of ablation behaviour, elements fall into several distinct clusters and that the elements within these clusters correlate well with each other during a period of ablation. Thus, elements within a cluster can be determined using an internal reference element from within the same cluster. While a combination of periodic varying properties typifies the clusters, the geochemical classification of elements into lithophile (silicate loving), and chalcophile (sulphide loving) appears to offer the best characterization of the major groups.

Journal Article↗
PubMed1995

Movement of the position of the transition state in protein folding.

Hammond behavior, in which two neighboring states move closer to each other along the reaction coordinate as the energy difference between them becomes smaller, has previously been observed for the transition state of unfolding of barnase. Here, we report Hammond behavior for the small protein chymotrypsin inhibitor 2 (CI2), which folds and unfolds via a single rate-determining transition state and simple two-state kinetics. Mutants have been generated along the entire sequence of the protein and the kinetics of folding and unfolding measured as a function of concentration of denaturant. The transition state was found to move progressively closer to the folded state on destabilization of the protein by mutation. Different regions of CI2 all show a similar sensitivity to changes in the energy of the transition state. This is in contrast to the behavior of barnase on mutation for which the position of the transition state for its unfolding is sensitive to mutation in some regions, especially in its major alpha-helix, but not in others. The transition state for the folding and unfolding of CI2 resembles an expanded version of the folded state and is formed in a concerted manner, in contrast to that for barnase, in which some regions of structure are fully formed and others fully unfolded. The reason for the general sensitivity of the position of the transition state of CI2 to mutation is presumably the relatively uniform degree of structure formation in the transition state and the concerted nature of its formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry↗
PubMed1994

Contribution of residues in the reactive site loop of chymotrypsin inhibitor 2 to protein stability and activity.

Residues in the active site loop of the serine protease inhibitor, chymotrypsin inhibitor 2, thought to play an important role in loop stability and inhibitory activity, have been investigated by site-directed mutagenesis. Substitutions at residues 58 (threonine in wild type) and 60 (glutamic acid in wild type), which flank the scissile bond (Met-59-Glu-60) and are conserved among the potato inhibitor I family of serine protease inhibitors, are found to be of some importance in the global stability of the protein, as measured by guanidinium chloride-induced denaturation, but are essential for its inhibitory activity. Mutation of either Thr-58 or Glu-60 to alanine results in a decrease in stability of 0.7 +/- 0.1 kcal mol-1. These values reflect the loss of hydrogen bonds between the hydroxyl group of Thr-58 with Glu-60 and Arg-67 and hydrogen bonds and a salt bridge between Glu-60 and Arg-62 and Arg-65. In addition, these mutants were found to be much weaker inhibitors of the serine protease subtilisin BPN'. The dissociation constants for inhibition, Ki, were found to be (7.0 +/- 0.4, 540 +/- 30, and 980 +/- 50) x 10(-13) M, for wild type, T58A, and E60A, respectively. Further, we find that these mutants are only temporary inhibitors of subtilisin BPN', unlike wild type. Over long time scales, we observe a reversal of inhibition because of hydrolysis of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding.

The equilibrium and kinetics of folding of the single-domain protein chymotrypsin inhibitor 2 conform to the simple two-state model. The structure of the rate-determining transition state has been mapped out at the resolution of individual side chains by using the protein engineering method on 74 mutants that have been constructed at 37 of the 64 residues. The structure contains no elements of secondary structure that are fully formed. The majority of interactions are weakened by > 50% in the transition state, although most regions do have some very weak structure. The structure of the transition state appears to be an expanded form of the native state in which secondary and tertiary elements have been partly formed concurrently. This is consistent with a "global collapse" model of folding rather than a framework model in which folding is initiated from fully preformed local secondary structural elements. This may be a general feature for the folding of proteins lacking a folding intermediate and is perhaps representative of the early stages of folding for multidomain or multimodule proteins. The major transition state for the folding of barnase, for example, has some fully formed secondary and tertiary structural elements in the major transition state, and barnase appears to form by a framework process. However, the fully formed framework may be preceded by a global collapse, and a unified folding scheme is presented.

Chymotrypsin↗
PubMed1993

Contribution of long-range electrostatic interactions to the stabilization of the catalytic transition state of the serine protease subtilisin BPN'.

The possible role of long-range electrostatic interactions on the catalytic activity of the serine protease subtilisin BPN' is investigated using protein engineering techniques. Charged residues on the surface of the enzyme some 13-15 A from the active site were mutated to either neutral or oppositely charged residues. The effect of these mutations on the stability of a complex formed between subtilisin BPN' and Z-Ala-Ala-Pro-Phe-trifluoromethyl ketone, a transition-state inhibitor of the enzyme, was measured. The values of Ki for the complex between the trifluoromethyl ketone and wild-type and mutant subtilisins were used to study the possible contribution of long-range electrostatics in stabilizing the charge distribution in the complex and thus, by analogy, on the transition state of hydrolysis for subtilisin BPN'. Measurement of kon, koff, and Ki for the inhibition of wild-type and mutant subtilisins showed that charged mutations distant from the active site can affect koff and Ki but have little effect on kon. The experimental results show that there is a small, 0.10-0.46 kcal mol-1, but significant contribution to the binding energy from distant surface charges, at low ionic strength. The experimental results were compared to theoretical results, calculated using the DelPhi program for different charge distributions in the complex. The experimental results were found to be most consistent with a complex in which an ion pair is formed between the protonated active site histidine and the ionized oxyanion. Both experimental and theoretical results suggest that long-range electrostatic interactions do play a role in stabilizing the transition-state complex formed between enzyme and inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1993

Effect of cavity-creating mutations in the hydrophobic core of chymotrypsin inhibitor 2.

Hydrophobic residues in the core of a truncated form of chymotrypsin inhibitor 2 (CI2) have been mutated in order to measure their contribution to the stability of the protein. The free energy of unfolding of wild-type and mutants was measured by both guanidinium chloride-induced denaturation and differential scanning calorimetry. The two methods give results for the changes in free energy on mutation that agree to within 1% or 2%. The average change in the free energy of unfolding (+/- standard deviation) for an Ile-->Val mutation is 1.2 +/- 0.1 kcal mol-1, for a Val-->Ala mutation 3.4 +/- 1.5 kcal mol-1, and for either an Ile-->Ala or a Leu-->Ala mutation 3.6 +/- 0.6 kcal mol-1. This gives an average change in the free energy of unfolding for deleting one methylene group of 1.3 +/- 0.5 kcal mol-1. Two significant correlations were found between the change in the free energy of unfolding between wild-type and mutant, delta delta GU-F, and the environment of the mutated residue in the protein. The first is between delta delta GU-F and the difference in side-chain solvent-accessible area buried between wild-type and mutant (correlation coefficient = 0.81, 10 points). The second and slightly better correlation was found between delta delta GU-F and N, the number of methyl/methylene groups within a 6-A radius of the hydrophobic group deleted (correlation coefficient = 0.84, 10 points). The latter correlation is very similar to that found previously for barnase, suggesting that this relationship is general and applies to the hydrophobic cores of other globular proteins. The combined data for C12 and barnase clearly show a better correlation with N (correlation coefficient = 0.87, 30 points) than with the change in the solvent-accessible surface area (correlation coefficient = 0.82, 30 points). This indicates that the packing density around a particular residue is important in determining the contribution the residue makes to protein stability. In one case, Ile-->Val76, a mutation which deletes the C delta 1 methyl group of a buried side chain, a surprising result was obtained. This mutant was found to be more stable than wild-type by 0.2 +/- 0.1 kcal mol-1. We have solved and analyzed the crystal structure of this mutant and find that there are small movements of side chains in the core, the largest of which, 0.7 A, is a movement of the side chain that has been mutated.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Structure of the hydrophobic core in the transition state for folding of chymotrypsin inhibitor 2: a critical test of the protein engineering method of analysis.

Chymotrypsin inhibitor 2 (CI2) unfolds and refolds according to a simple two-state kinetic mechanism. The single rate-determining transition state may thus be studied by kinetics of both unfolding and refolding. This has allowed the direct testing of some facets of the protein engineering procedure (phi-value analysis). The structure of the hydrophobic core of CI2 in the transition state was analyzed from kinetic and thermodynamic measurements of guanidinium chloride-induced unfolding of 11 mutants and of their rates of refolding. In all cases, the strengths of the interactions measured from refolding kinetics in water are in excellent agreement with those measured from unfolding kinetics in guanidinium chloride solutions and extrapolated to zero molar denaturant. Changes in the free energies of unfolding on mutation, as well as other equilibrium properties calculated from the rate constants, are also in excellent agreement with those measured directly from equilibrium studies. These data provide further evidence for application of the principle of microscopic reversibility to aspects of protein folding in the presence of denaturant and the validity of extrapolation to the absence of denaturant. The edges of the hydrophobic core of CI2 are significantly weakened in the transition state, and, in many cases, the interactions are totally lost. The center of the core remains partially intact; the interaction energy is lowered by about 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition.

The reversible folding and unfolding of barley chymotrypsin inhibitor 2 (CI2) appears to be a rare example in which both equilibria and kinetics are described by a two-state model. Equilibrium denaturation by guanidinium chloride and heat is completely reversible, and the data can be fitted to a simple two-state model involving only native and denatured forms. The free energy of folding in the absence of denaturant, delta GH2O, at pH 6.3, is calculated to be 7.03 +/- 0.16 and 7.18 +/- 0.43 kcal mol-1 for guanidinium chloride and thermal denaturation, respectively. Scanning microcalorimetry shows that the ratio of the van't Hoff enthalpy of denaturation to the calorimetric enthalpy of denaturation does not deviate from unity, the value observed for a two-state transition, over the pH range 2.2-3.5. The heat capacity change for denaturation is found to be 0.789 kcal mol-1 K-1. The rate of unfolding of CI2 is first order and increases exponentially with increasing guanidinium chloride concentration. Refolding, however, is complex and involves at least three well-resolved phases. The three phases result from heterogeneity of the unfolded form due to proline isomerization. The fast phase, 77% of the amplitude, corresponds to the refolding of the fraction of the protein that has all its prolines in a native trans conformation. The rate of this major phase decreases exponentially with increasing guanidinium chloride concentration. The unfolding and refolding kinetics can also be fitted to a two-state model.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry↗