SAXS models of TGFBIp reveal a trimeric structure and show that the overall shape is not affected by the Arg124His mutation.

Human transforming growth factor ß induced protein (TGFBIp) is composed of 683 residues, including an N-terminal cysteine-rich (EMI) domain, four homologous fasciclin domains, and an Arg-Gly-Asp (RGD) motif near the C-terminus. The protein is of interest because mutations in the TGFBI gene encoding TGFBIp lead to corneal dystrophy (CD), a condition where protein aggregates within the cornea compromise transparency. The complete three-dimensional structure of TGFBIp is not yet available, with the exception of a partial X-ray structure of the archetype FAS1 domain derived from Drosophila fasciclin-1. In this study, small-angle X-ray scattering (SAXS) models of intact wild-type (WT) human TGFBIp and a mutant (R124H) are presented. The mutation R124H leads to a variant of granular CD. The deduced structure of the TGFBIp monomer consists of four FAS1 domains in a simple "beads-on-a-string" arrangement, constructed by the superimposition of four consecutive Drosophila fasciclin domains. The SAXS-based model of the TGFBIp R124H mutant displayed no structural differences from WT. Both WT TGFBIp and the R124H mutant formed trimers at higher protein concentrations. The similar association properties and three-dimensional shape of the two proteins suggest that the mutation does not induce any major structural rearrangements, but points towards the role of other corneal-specific factors in the formation of corneal R124H deposits.

Modafinil for attentional and psychomotor dysfunction in advanced cancer: a double-blind, randomised, cross-over trial.

Cognitive impairment seems to be highly prevalent in patients with advanced cancer. Modafinil, a novel vigilance and wake-promoting agent, may be an alternative treatment. We wanted to investigate this treatment on attentional and psychomotor dysfunction in cancer patients. 28 cancer patients with a tiredness score of 50 mm or more on a scale of 0 to 10 (0=no tiredness, 10=worst possible tiredness) and Karnofsky Performance Status 40-70 were included. All medications were kept stable during the trial despite short acting opioids for breakthrough pain. On day 1 the patients were randomly assigned to receive 200 mg Modafinil orally or placebo and on day 4 they crossed-over to the alternative treatment. Finger Tapping Test (FTT), Trail Making Test (TMT) and Edmonton Symptom Assessment System (ESAS) were evaluated before tablet intake and again 4, 5 hours after. FTT for the dominant hand as well as TMT were statistically significantly improved on modafinil (p-values=0.006 and 0.042, respectively). On ESAS, depression and drowsiness also improved statistically significantly (p-values=<0.001 and 0.038, respectively). Modafinil in a single dose regimen was significantly superior to placebo regarding two cognitive tests of psychomotor speed and attention. Furthermore subjective scores of depression and drowsiness were significantly improved by modafinil.

Motilin- and neuropeptide Y-like immunoreactivity in a psychophysiological stress experiment on patients with functional dyspepsia.

BACKGROUND: The aim of the study was to compare plasma motilin-like immunoreactivity (MOT-LI) and neuropeptide Y (NPY)-like immunoreactivity (NPY-LI) in patients with functional dyspepsia (FD) during a controlled psychophysiological experiment. METHOD: 25 patients (12 men, 13 women), age 24-50, with recurrent FD, and 25 pair-wise sex- and age-matched community control subjects were studied. In an experiment, after a rest period, subjects were studied during a 15-min stress interview. The aim of the interview was to elicit anxiety. Before and during the intervention blood samples were drawn for peptide analyses. Outcome measures were the Gastrointestinal Symptom Rating Scale, fasting blood glucose, heart rate and blood pressure as well as the subjects' self-ratings on visual analogue scales. The plasma concentrations of MOT-LI and NPY-LI are given as anti-logarithms. RESULTS: Mean plasma MOT-LI concentration was 7.3 (CI: 5.7-9.4) pmol/L in the patient group, and 7.9 (CI: 6.1-10.2) pmol/L in the control group. Mean plasma NPY-LI concentration was 14.2 (CI: 12.3-16.4) pmol/L in the patient group, and 13.4 (Cl: 11.8-15.3) pmol/L in the control group. Using ANCOVA (covariates: group, gender, age, body mass index and smoking) MOT-LI was related to lower indigestion symptomatology (p<0.04) and positive change in joyfulness during the interview (p<0.03). In the patient group delta motilin correlated with increased joyfulness (p<0.03) and decreased sadness (p<0.03). The NPY-LI increase during the interview was related to higher fasting blood glucose before the interview (p<0.01) and a stronger increase in systolic blood pressure during the test (p<0.05). CONCLUSION: During a stress interview plasma MOT-LI is positively related to less indigestion symptomatology and joyfulness, while changes in plasma NPY-LI were positively related to sympathetic nervous system activity, but not to gastrointestinal symptoms.

Structural mapping of an aggregation nucleation site in a molten globule intermediate.

Protein aggregation plays an important role in biotechnology and also causes numerous diseases. Human carbonic anhydrase II is a suitable model protein for studying the mechanism of aggregation. We found that a molten globule state of the enzyme formed aggregates. The intermolecular interactions involved in aggregate formation were localized in a direct way by measuring excimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. The contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large beta-sheet of the protein, within a limited region between the central beta-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific beta-sheet containing regions in aggregate formation.

N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.

Low plasma prolactin levels in patients with functional dyspepsia.

BACKGROUND: The purpose was to compare the levels and patterns of plasma cortisol and prolactin in patients with functional dyspepsia (FD) during a controlled laboratory experiment. METHOD: 25 patients (12 men, 13 women), aged 24-50, with recurrent FD, and 25 pair-wise gender- and age-matched community control subjects were studied at a gastroenterological laboratory in a Swedish university hospital. In an experiment, after a rest period, subjects were studied during a neutral interview and a stress interview. Before and during interventions, blood samples were drawn for later peptide analyses. The main outcome measures were the Gastrointestinal Symptom Rating Scale, heart rate, blood pressure, plasma cortisol and prolactin. RESULTS: Mean plasma cortisol concentration correlated negatively with diarrhoea symptoms (partial correlation; p < 0.01). The level of plasma proclatin ( microg/l) was significantly lower (paired t test; p < 0.01) in the patient group (mean = 3.34, CI: 2.75-3.93) compared to the control group (mean = 4.70, CI: 3.63-5.78). During the stress interview, prolactin increased significantly in both groups. When the whole sample was divided according to degree of reflux symptoms, those with high reflux symptomatology had lower prolactin (ANCOVA with covariates for group, gender, age, body mass index and smoking; p < 0.05). CONCLUSION: Plasma prolactin concentration was significantly lower in FD patients compared to a matched control group. A high degree of reflux symptoms was significantly associated with inhibition of the prolactin increase during a stress interview.

Denatured states of human carbonic anhydrase II: an NMR study of hydrogen/deuterium exchange at tryptophan-indole-H(N) sites.

Hydrogen/deuterium (H/D) exchange measurements in low and moderate concentrations of GuHCI were conducted on the side chain H(N) atoms of the seven tryptophans of pseudo wild-type human carbonic anhydrase II. Tryptophans 5, 16 and 245, situated in or close to the N-terminal domain were found to have little protection against exchange. The H/D exchange results for Trp-123, Trp-192 and Trp-209 showed that a previously identified molten globule and the native state gave a similar protection against exchange. Global unfolding of the protein is necessary for the efficient exchange at Trp-97, which is located in the central part of the beta-sheet.

Hydration of denatured and molten globule proteins.

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of alpha-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.

EPR mapping of interactions between spin-labeled variants of human carbonic anhydrase II and GroEL: evidence for increased flexibility of the hydrophobic core by the interaction.

Human carbonic anhydrase II (HCA II) interacts weakly with GroEL at room temperature. To further investigate this interaction we used electron paramagnetic resonance (EPR) spectroscopy to study HCA II cysteine mutants spin-labeled at selected positions. From our results it is evident that protein-protein interactions can be specifically mapped by site-directed spin-labeling and EPR measurements. HCA II needs to be unfolded to about the same extent as a GuHCl-induced molten-globule intermediate of the enzyme to interact with GroEL. The interaction with GroEL includes interactions with outer parts of the HCA II molecule, such as peripheral beta-strands and the N-terminal domain, which have previously been shown to be rather unstable. As a result of the interaction, the rigid and compact hydrophobic core exhibits higher flexibility than in the molten globule, which is likely to facilitate rearrangements of misfolded structure during the folding process. The degree of binding to GroEL and accompanying inactivation of the enzyme depend on the stability of the HCA II variant, and nonspecific hydrophobic interactions appear to be most important in stabilizing the GroEL-substrate complex.

Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide.

The crystal structure of carbonic anhydrase from Neisseria gonorrhoeae has been solved to a resolution of 1.78 A by molecular replacement using human carbonic anhydrase II as a template. After refinement the R factor was 17.8% (Rfree=23.2%). There are two molecules per asymmetric unit (space group P21), but they have essentially identical structures. The fold of the N. gonorrhoeae enzyme is very similar to that of human isozyme II; 192 residues, 74 of which are identical in the two enzymes, have equivalent positions in the three-dimensional structures. This corresponds to 85% of the entire polypeptide chain of the bacterial enzyme. The only two cysteine residues in the bacterial enzyme, which has a periplasmic location in the cell, are connected by a disulfide bond. Most of the secondary structure elements present in human isozyme II are retained in N. gonorrhoeae carbonic anhydrase, but there are also differences, particularly in the few helical regions. Long deletions in the bacterial enzyme relative to human isozyme II have resulted in a considerable shortening of three surface loops. One of these deletions, corresponding to residues 128 to 139 in the human enzyme, leads to a widening of the entrance to the hydrophobic part of the active site cavity. Practically all the amino acid residues in the active site of human isozyme II are conserved in the N. gonorrhoeae enzyme and have similar structural positions. However, the imidazole ring of a histidine residue, which has been shown to function as a proton shuttle in the catalytic mechanism of the human enzyme, interacts with an extraneous entity, which has tentatively been identified as a 2-mercaptoethanol molecule from the crystallization medium. When this entity is removed by soaking the crystal in a different medium, the side-chain of His66 becomes quite mobile. The structure of a complex with the sulfonamide inhibitor, acetazolamide, has also been determined. Its position in the active site is very similar to that observed in human carbonic anhydrase II.

Gastrin, cholecystokinin, and somatostatin in a laboratory experiment of patients with functional dyspepsia.

OBJECTIVE: To explore the pattern of gastrointestinal hormonal variations in plasma and to relate this to possible pathophysiological mechanisms in functional dyspepsia. METHOD: There were 25 patients, 12 men and 13 women, aged 24 to 50 years, with recurrent functional dyspepsia, compared with community control subjects pair-wise, matched for age and sex. The subjects participated in a laboratory stress experiment with timed provocations. At fixed intervals, 22 samples of blood were drawn from each subject and frozen for later peptide analyses. Levels of gastrin, cholecystokinin (CCK), and somatostatin were measured by radioimmunoassay. Peptide levels were studied during a friendly greeting, a stress interview, and a food stimulation. RESULTS: Mean hormone values did not differ between the groups. Smokers had lower mean CCK than nonsmokers. Patients with a high degree of dyspeptic symptoms during the week preceding the experiment had a higher mean somatostatin level than patients with a low degree of dyspeptic symptoms. Heartburn correlated positively with the mean somatostatin level. Mean gastrin correlated with body mass index. During the 15-minute stress interview, significant changes in peptide variations were noted: Gastrin increased in both patient and control group subjects. CCK levels increased in patients from 7.2 pmol/l (6.0-8.5) to 9.8 pmol/l (8.2-11.4), but not in control subjects (p < 0.04, two-way interaction). Somatostatin increased significantly earlier in patients than in the control subjects during the stress interview. CONCLUSIONS: A positive relationship was found between the mean level of somatostatin and the degree of dyspeptic symptoms. Gastrin, CCK, and somatostatin were all sensitive to an anxiety-provoking interview. CCK and somatostatin may possibly link psychological reactions to the pathophysiology of functional dyspepsia.

Preliminary NMR studies of Thermus thermophilus ribosomal protein S19 overproduced in Escherichia coli.

The gene for the ribosomal protein S19 from Thermus thermophilus was cloned, sequenced and overexpressed in Escherichia coli. A simple procedure for isolating the recombinant protein was developed. Preliminary NMR studies revealed a high content of alpha-helical secondary structure in the protein.

A mutant form of the ribosomal protein L1 reveals conformational flexibility.

The crystal structure of the mutant S179C of the ribosomal protein L1 from Thermus thermophilus has been determined at 1.9 A resolution. The mutant molecule displays a small but significant opening of the cavity between the two domains. The domain movement seems to be facilitated by the flexibility of at least two conserved glycines. These glycines may be necessary for the larger conformational change needed for an induced fit mechanism upon binding RNA. The domain movement makes a disulfide bridge possible between the incorporated cysteines in two monomers of the mutant L1.

Remarkably slow folding of a small protein.

Equilibrium denaturation of the 72 amino acid alpha/beta-protein MerP, by acid, guanidine hydrochloride, or temperature, is fully reversible and follows a two-state model in which only the native and unfolded states are populated. A cis-trans equilibrium around a proline peptide bond causes a heterogeneity of the unfolded state and gives rise to a slow- and a fast folding population. With a rate constant of 1.2 s(-1) for the major fast folding population, which has none of the common intrinsically slow steps, MerP is the slowest folding protein of this small size yet reported.

Formation of local native-like tertiary structures in the slow refolding reaction of human carbonic anhydrase II as monitored by circular dichroism on tryptophan mutants.

In the present study, near-UV CD kinetic measurements on mutants, in which one Trp residue had been replaced, were performed to probe the development of asymmetric environments around specific Trp residues during the refolding of human carbonic anhydrase II (HCAII). In addition, the formation of the active site was probed by the binding of a fluorescent sulfonamide inhibitor. The development of the individual Trp CD spectra during refolding was obtained by subtracting the CD spectrum of the mutant lacking one Trp from that of HCAII at different time points. The same method was used for the particular Trp residues to obtain the kinetic CD traces monitored at a specific wavelength (270 nm). Trp residues 16, 97, and 245 were analyzed. Trp16 probes the N-terminal domain (amino acid residues 1-25), and this part is forming its tertiary structure slower than the major domain (amino acid residues 26-260) of the protein molecule, which contains the active site and a dominating beta-sheet. An essentially native structure of the major domain seems to act as a template for the correct folding of the N terminus. Trp97 is located in a hydrophobic cluster comprising beta-strands 3-5 in the protein core. Previously, we have shown that this region is remarkably stable and compact, and stopped-flow fluorescence data indicate that Trp97 is buried in an apolar compact cluster within a few milliseconds [Svensson, M., Jonasson, P., Freskgård, P.-O., Jonsson, B.-H., Lindgren, M., Martensson, L.-G., Gentile, M., Bóren, K., & Carlsson, U. (1995) Biochemistry 34, 8606-8620; Jonasson, P., Aronsson, G., Carlsson, U., & Jonsson, B.-H. (1997) Biochemistry 36 (in press)]. Here it is shown that the development of the native tertiary structure at Trp97 occurs in the minute time domain. Trp245 is located in a long loop between the N-terminal domain and the core structure. Although this Trp has attained native-like fluorescence properties within the dead time of the CD experiment, it assumes a native-like asymmetric environment even slower than Trp97. Thus, the investigated Trp residues develop their native CD bands at different rates, showing that formation of native-like tertiary structure is occurring with varying rates in different regions of the protein.

Tertiary structure formation at specific tryptophan side chains in the refolding of human carbonic anhydrase II.

The refolding reaction of human carbonic anhydrase II has been characterized by use of seven variants in which tryptophan residues have been replaced by Phe or Cys, in each case giving proteins with six tryptophans. Intrinsic tryptophan fluorescence was used to monitor the refolding in the 2 ms-60 s time range, and kinetic traces showing the contributions from each particular tryptophan were obtained by calculation of differences between the wild-type protein and the variants. Earlier assignment [Mårtensson, L.-G., Jonasson, P., Freskgard, P.-O., Svensson, M., Carlsson, U., & Jonsson, B.-H. (1995) Biochemistry 34, 1011-1021] of specific fluorescence properties to each tryptophan, especially regarding energy transfer and intrinsic fluorescence quenching, has made it possible to use the kinetic data to describe the formation of tertiary structure at defined tryptophan residues. In summary, it was found that tertiary structure is formed earlier at those tryptophans that are associated with the central core of beta-strands than at tryptophan residues in the N-terminal minidomain.

The complete sequence, expression in Escherichia coli, purification and some properties of carbonic anhydrase from Neisseria gonorrhoeae.

The complete nucleotide sequence of the carbonic anhydrase gene from Neisseria gonorrhoeae has been determined. The gene encodes a 252-residue polypeptide with a molecular mass of 28085 Da. The gene has been cloned and overexpressed in Escherichia coli, and the enzyme has been purified. A 26-residue signal peptide is cleaved off by the E. coli processing machinery. Thus, the isolated enzyme contains 226 amino acid residues with a molecular mass of 25314 Da. Most of the enzyme seems to be produced as a soluble protein located in the periplasm of E. coli. The enzyme is homologous to carbonic anhydrases from the animal kingdom; it is an alpha-carbonic anhydrase. A comparison with the amino acid sequences of human carbonic anhydrases I and II suggests that the secondary structures are essentially intact in the bacterial enzyme but that several loops are much shorter than in the human forms. Most of the active-site residues are identical to those found in the high-activity human isozyme II. The bacterial enzyme has a high CO2 hydration activity with a k(cat) of 1.1 x 10(6) s(-1) and Km of 20 mM at pH 9 and 25 degrees C. The enzyme also catalyzes the hydrolysis of 4-nitrophenyl acetate. The pH/rate profile can be described as a titration curve with pKa of 6.7 and a maximal value of the catalytic second-order rate constant, k(enz), of 130 M(-1) x s(-1).

Adsorption to silica nanoparticles of human carbonic anhydrase II and truncated forms induce a molten-globule-like structure.

Human carbonic anhydrase II pseudo-wild type (HCAIIpwt) and two truncated variants were adsorbed to approximately 9 nm silica nanoparticles. Ellipsometry was used as an indirect measure of protein adsorption. The structural changes of adsorbed proteins were investigated with the use of circular dichroism (CD), intrinsic fluorescence, ANS binding ability and inhibitor binding capacity. It was found that the variants that were truncated at positions 5 and 17 in the N-terminal end attain a molten-globule-like state after interaction with the silica nanoparticles. In contrast, the more stable HCAIIpwt retained most of its native structure after 24 h adsorption to silica nanoparticles. The result suggests that surface induced unfolding may give rise to intermediates similar to those for unfolding induced by, for example GuHCl. Thus, the intermediate observed has some features of the molten globule.