Fourier-transform infrared spectroscopy (FTIR) has been employed to investigate the structural properties of the 28-kDa channel-forming integral membrane protein (CHIP28) present in phospholipid vesicles suspended in aqueous media. This study reports the FTIR spectra of this membrane protein present in H2O and 2H2O. The secondary structure of the protein was determined and found to consist of 36% alpha-helical and 42% beta-sheet structures. These results are in close agreement with the results of a previous CD study [Van Hoek, A. N., Wiener, M., Bicknese, S., Miercke, L., Biwersi, J. & Verkman, A. S. (1993) Biochemistry 32, 11,847-11,856]. However, the results differ from those given in an FTIR analysis by the same workers who recorded FTIR spectra of the CHIP28 protein in a dehydrated state. An unusually high extent of hydrogen-deuterium exchange of the peptide groups of this protein occurs. The magnitude of the spectral changes observed upon exposure of the protein to 2H2O is greater than has been observed with any other membrane protein previously studied. Thus, over 80% of the peptide groups exchange within 5 min and the amide I band maximum shifts to low frequency by approximately 20 cm-1. This high hydrogen-deuterium exchange observed with the CHIP28 protein is consistent with the presence of an aqueous pore within the protein structure.

Aquaporin 1↗

PubMed1995

Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.

Combinatorial mismatch-primer mutagenesis was used to make simultaneous changes of codons for residues Trp19 and Trp99 of the Escherichia coli trp aporepressor (TrpR protein) to codons for other residues. Among 21 different single- and double-mutant repressors obtained from this round of mutagenesis, proteins with Trp-->Leu and Trp-->Met changes at one or both positions were found to be nearly as active as the wild type (wt). Genes encoding repressors with each of the eight possible combinations of single- and double-mutant changes of Trp19 and Trp99 to Leu and Met were constructed by recombination in vitro. Whereas three of these eight mutant repressors are unstable in E. coli, all are made at similar steady-state levels in Salmonella typhimurium. Three of the eight mutant holorepressors are lethal when overproduced in S. typhimurium, because they confer an induced auxotrophy. Two different activity assays in vivo show that one of the four double-mutant repressors (Trp19-->Leu; Trp99-->Met) is similar to wt TrpR in its interactions with both Trp and DNA. These results show that more general approaches to engineering active proteins with fewer Trp residues may give rise to functional mutants without aromatic substitutions, and that aliphatic changes should be considered in cases where engineered changes of Trp to Phe or Tyr do not work.

Amino Acid Sequence↗

PubMed1995

Temperature-induced changes in protein structures studied by Fourier transform infrared spectroscopy and global analysis.

Fourier transform infrared (FTIR) spectroscopy has been used to study temperature-induced structural changes which occur in albumin, immunoglobulin G, fibrinogen, lysozyme, alpha-lactalbumin, and ribonuclease S when dissolved in 2H2O. In order to analyze the data, a new method was developed in which the data were analyzed globally with the aid of a spectral model. Seven or eight bands were sufficient to fit the full data set of spectra ranging from 1420 to 1760 cm-1 with a root mean square error of 1-2% of the maximum. Subsequently, the estimated band amplitude curves which showed a sigmoidal progression with increasing temperature were (globally) fitted with a two-state thermodynamic model. In this way, information on structural changes as well as on the thermal stability of the proteins was obtained. In all proteins investigated, enhanced 1H-2H exchange occurred at temperatures well below the unfolding of the secondary structure. This was interpreted as a change in tertiary structure leading to enhanced solvent accessibility. In all the proteins investigated, except for ribonuclease S, an intermolecular beta-sheet band indicative of aggregation appeared concomitant with the denaturation of the secondary structure. The results are compared with data from other techniques and discussed in terms of local unfolding and folding intermediates.

Animals↗

PubMed1995

Balanced reciprocal translocation mosaicism: how frequent?

We describe 2 cases of balanced reciprocal translocation (BRT) mosaicism. The frequency of this aberration in the population referred to our laboratory was determined and compared to those frequencies reported in the literature by other clinical cytogenetics laboratories. The extent of BRT mosaicism was also examined in surveys of parental populations, which are less likely to have a bias due to ascertainment on the basis of abnormal phenotype. the frequencies in the postnatal and prenatal populations examined in this study were calculated to be 5.7 x 10(-5) (95% confidence interval is 3.2-8.2 x 10(-5)) and 4.1 x 10(-5) (95% confidence interval is 2.0-6.2 x 10(-5)). However, in view of the extent of variation reported in the various studies, these estimates should be considered first approximations of the true frequency.

Abnormalities, Multiple↗

PubMed1995

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems.

FT-IR spectroscopy has been applied to study the secondary structure of the major coat protein of Pf1 and M13 as present in the phage and reconstituted in DOPG and mixed DOPC/DOPG (4/1) bilayers. Infrared absorbance spectra of the samples were examined in dehydrated films and in suspensions of D2O and H2O. The secondary structure of the coat protein is investigated by second-derivative analysis, Fourier self-deconvolution, and curve fitting of the infrared bands in the amide I region (1600-1700 cm-1). It is found that, in dehydrated films of Pf1 and M13 phage, the amide I region contains three bands located at about 1633, 1657, and 1683 cm-1, that are assigned to hydrogen-bonded turn, alpha-helix/random coil, and non-hydrogen-bonded turn, respectively. From a comparison of the infrared spectra in dehydrated film with those in aqueous suspension, the percentages of secondary structure were found with an accuracy of about +/- 5%. For the coat protein of Pf1 phage, the FT-IR quantification gives 69% alpha-helix conformation, 19% turn structure, and 12% random coil structure. For Pf1 coat protein in the membrane-embedded state, the amount of alpha-helix is 57%, whereas 42% is in a turn structure and 1% in a random coil structure. The same assignment strategy was used for the analysis of the data obtained for M13 coat protein reconstitution into phospholipid systems. For M13 coat protein in the phage, this gives 75% alpha-helix conformation, 21% turn structure, and 4% random coil structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗

PubMed1995

A comparison of infrared spectra of proteins in solution and crystalline forms.

Fourier transform infrared spectroscopy has been used to compare the structure of a range of proteins in solution and in the form of single crystals. An infrared microscope was used to record the spectra of single crystals of the proteins. The proteins studied in this way were hen egg white lysozyme, bovine pancreatic ribonuclease A, bovine gamma-II crystallin, human serum amyloid P component, Endothia parasitica pepsin and Mucor pusillus pepsin. The amide I and amide II bands in the FTIR spectra of these proteins were analysed using derivative procedures thereby providing information on the secondary structure. The crystals were held under a vapour of mother liquor to reduce the effects of dehydration. A comparison of the spectra revealed that spectra recorded from crystals of lysozyme, ribonuclease A and gamma-II crystallin are nearly identical to those recorded from the proteins in solution. However, differences are observed between the spectra of serum amyloid P component, Endothia parasitica pepsin and Mucor pusillus pepsin in solution compared with that of the crystalline form These differences are suggested to be due to rearrangements of turn structures within the protein structure.

Animals↗

PubMed1995

The conformational analysis of peptides using Fourier transform IR spectroscopy.

Fourier transform infrared spectroscopy (FTIR) can be used for conformational analysis of peptides in a wide range of environments. Measurements can be performed in aqueous solution, organic solvents, detergent micelles as well as in phospholipid membranes. Information on the secondary structure of peptides can be derived from the analysis of the strong amide I band. Orientation of secondary structural elements within a lipid bilayer matrix can be determined by means of polarized attenuated total reflectance-FTIR spectroscopy. Hydrogen-deuterium exchange can be monitored by the analysis of the amide II band. This review gives some example of peptide systems studied by FTIR spectroscopy. Studies on alamethicin and alpha-aminoisobutyric acid containing peptides have shown that FTIR spectroscopy is a sensitive tool for identifying 3(10)-helical structures. Changes in the structure of the magainins upon interaction with charged lipids were detected using FTIR spectroscopy. Tachyplesin is an example of a beta-sheet containing membrane active peptide. Polarized ir spectroscopy reveals that the antiparallel beta-sheet structures of tachyplesin are oriented parallel to the membrane surface. Synthesis of peptides corresponding to functionally/structurally important regions of large proteins is becoming increasingly popular. FTIR spectroscopy has been used to analyze the structure of synthetic peptides corresponding to the ion-selective pore of the voltage-gated potassium channel. In biomembrane systems these peptides adopt a highly helical structure. Under conditions, where these peptides are aggregated the presence of some intermolecular beta-sheet structure can also be detected.

Alamethicin↗

PubMed1995

Plasmodium berghei infection: dichloroacetate improves survival in rats with lactic acidosis.

The kinetics of Plasmodium berghei infection and the development of lactic acidosis, hypoglycemia, and anemia were defined in young Wistar rats. This model of metabolic dysfunction, which is similar to that of severe human malaria, was used to test the hypothesis that dichloroacetate, a treatment for lactic acidosis, prolonged survival in rats receiving a single antimalarial dose of quinine (20 mg/kg). Rats with hyperlactatemia (lactate > 5 mmol/liter, N = 183) were randomized to receive either dichloroacetate (100 mg/kg, N = 99) or saline (N = 84) and were monitored for outcome (survival or death) for 50 hr. Logistic regression modeling adjusting for baseline venous lactate concentration demonstrated that dichloroacetate increases survival rates in rats with venous lactate concentrations between 5 and 8.9 mmol/liter (odds ratio > 2.2, P < 0.021). This is the first demonstration that specific intervention to treat lactic acidosis can prolong survival and suggests that dichloroacetate may be useful as adjunctive therapy in the management of lactic acidosis complicating severe falciparum malaria.

Acidosis, Lactic↗

PubMed1995

Navigator: tools for informal structure-activity relationship discovery.

Navigator is a molecular database visualization system, designed to support exploratory data analysis and informal structure-activity relationship studies. In addition to the operations commonly found in chemical database systems, it provides new tools that facilitate substituent analysis and help elucidate the relationships among similar molecules and between related assays. Navigator's capabilities include two ways of displaying the relationships between analogs, mouse-sensitive charts of sets of molecules, mouse-sensitive plots of assay relationships, and access to a system for three-dimensional quantitative structure-activity relationship discovery. Navigator's mouse-based user interface provides a one-object/one-window paradigm that makes data manipulation easy even for inexperienced users. Navigator runs on Silicon Graphics workstations.

Computer Graphics↗

PubMed1995

Structural characterisation of a slowly activating potassium channel (IsK).

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1995

Application of SPR & FTIR spectroscopy to the study of protein-biomaterial interactions.

Explore the source record for details and available documents.

Animals↗

PubMed1995

Clinical features and outcome of severe malaria in Gambian children.

The clinical and laboratory features of severe falciparum malaria in 180 Gambian children were studied between 1985 and 1989. Of the 180 children, 118 (66%) presented with seizures, 77 (43%) had cerebral malaria, 35 (20%) had witnessed seizures after admission, 29 (16%) were hypoglycemic, and 27 (15%) died. Respiratory distress was a common harbinger of a fatal outcome. The differences in admission parasite counts in the blood, hematocrit, and opening cerebrospinal pressures for patients who died and survivors were not significant. A multiple logistic regression model identified neurological status (coma, particularly if associated with extensor posturing), stage of parasite development on the peripheral blood film, pulse rate of > 150 or respiratory rate of > 50, hypoglycemia, and hyperlactatemia (plasma lactate level, > 5 mmol/L) as independent indicators of a fatal outcome. Biochemical evidence of hepatic and renal dysfunction was an additional marker of a poor prognosis, but, in contrast to severe malaria in adults, none of these children with severe malaria had acute renal failure.

Adult↗

PubMed1994

A spectroscopic study of the mitochondrial transit peptide of rat malate dehydrogenase.

A peptide corresponding to the N-terminal sequence of the rat malate dehydrogenase, comprising the transit sequence and two residues of the mature protein (MLSALARPVGAALR-RSFSTSAQNNAK) has been chemically synthesized, and its structural characteristics investigated by Fourier-transform i.r. (FT-IR), c.d. and 1H-n.m.r. spectroscopy. FT-IR and c.d. spectra of the peptide were recorded in a variety of environments (aqueous solution, trifluoroethanol) and after incorporation into phospholipid bilayers. The peptide was found to be mainly in aperiodic or undefined conformation in aqueous solution. However, in trifluoroethanol a marked increase in alpha-helical content was observed. An increase in alpha-helical content was also observed in negatively charged lipids (dimyristoylphosphatidylglycerol and cardiolipin). However, when reconstituted in a zwitterionic phospholipid (dimyristoylphosphatidylcholine), no alpha-helical structure was observed. N.m.r. spectroscopy was used to characterize the helical structure in greater detail in trifluoroethanol. The 1H-n.m.r. spectrum of the peptide in this solvent was assigned using standard homonuclear two-dimensional methods. The observed patterns of nuclear Overhauser enhancements confirmed the deductions obtained from c.d. and FT-1R spectroscopy concerning the solution conformation, suggesting a region of flexible nascent helix between Ala-4 and Ser-18. This structure is discussed in terms of the possible function of the peptide.

Alanine↗

PubMed1994

Structure and thermal stability of the extracellular fragment of human transferrin receptor at extracellular and endosomal pH.

Fourier transform infrared spectroscopy has been used to study the solution structure and thermal stability of the extracellular fragment of human transferrin receptor (tfRt) at extracellular and endosomal pH. At extracellular pH tfRt is composed of 56% alpha-helix, 19% beta-sheet and 14% turns. Upon acidification to endosomal pH the alpha-helical content of the protein is reduced and the beta-sheet content increased by nearly 10%. At extracellular pH, the midpoint temperature of thermal denaturation (Tm) for the loss of secondary and tertiary structure, and the formation of aggregated structures, is 71 degrees C. At endosomal pH this temperature is reduced by approximately 15 degrees C. The apparent entropies of thermal denaturation indicate that the native structure of tfRt at endosomal pH is far more flexible than at extracellular pH.

Endosomes↗