| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute of Protein Biochemistry and Enzymology, National Research Council, 80125 Naples, Italy
2 Department of Biochemistry and Biophysics, Second University of Naples, 80131 Naples, Italy
3 Departments of Chemistry and
4 Physics, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel
Reprint requests to: Roberto Nucci, Institute of Protein Biochemistry and Enzymology, National Research Council, Via Marconi 10 80125 Napoli, Italy; e-mail: nucci{at}dafne.ibpe.na.cnr.it; fax: +39/081 7257300.
(RECEIVED April 25, 2002; FINAL REVISION August 5, 2002; ACCEPTED August 5, 2002)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0212802.
 |
Abstract |
|---|
 TOP Abstract IntroductionResultsDiscussionConclusionMaterials and methodsReferences |
|---|
c) ranged from 6 x 107 to 2 x 108 sec-1 for the protein labeled at position C344 and from 5.62 x 107 to 1.10 x 108 sec-1 for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (c) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix. Keywords: Dynamics; chemical modification; mutant; electron paramagnetic resonance; frequency domain fluorometry
Abbreviations: Sßgly, Sulfolobus solfataricus ß-glycosidase • EcSßgly, Sulfolobus solfataricus ß-glycosidase expressed in E. coli • MAR•, Maleimido nitroxide radical • EcSßgly_S101C, mutant protein at position 101 in which serine was substituted with cysteine • EcSßgly-MAR•, EcSßgly labeled by MAR• • EcSßgly_S101C-MAR•, mutant protein labeled by MAR• • EPR, electron paramagnetic resonance • ONPG, o-nitro-phenyl-ß-D-galactopyranoside • PNPG, p-nitro-phenyl-ß-D-glucopyranoside • DTT, dithioerythrytol • DTNB, 5,5'-dithio-bis (2-nitrobenzoic acid)
|
Introduction |
|---|
|
TOPAbstractIntroductionResultsDiscussionConclusionMaterials and methodsReferences |
|---|
This paper examines the conformational flexibility/rigidity of ß-glycosidase from S. solfataricus by both spin labeling electron paramagnetic resonance (EPR) and by frequency domain fluorometry. To perform EPR investigations, the protein was labeled at Cys-344 and at Cys-101, in a mutant in which Ser-101 was changed into cysteine. Labels bound to a certain site of the protein provide local information about the occurrence in the vicinity (Likhtenshtein 1976Likhtenshtein 1993). Nevertheless, the labeling of biological objects by nitroxide spin labels proves highly useful, as the rotational correlation time of the bound spin label undergoes changes that reflect even the slightest modifications in the protein structure.
Time-resolved fluorescence techniques are powerful tools for studying the structural and dynamic aspects of protein macromolecules (Beechem and Brand 1985; Alcalà et al. 1987). These allow the analysis of emission decay arising from a great number of emitting species (e.g ß-glycosidase), which possesses 17 tryptophans for each subunit homogeneously dispersed in the primary structure, thus making it possible to investigate the dynamic behavior of the whole protein macromolecule.
|
Results |
|---|
|
TOPAbstractIntroductionResultsDiscussionConclusionMaterials and methodsReferences |
|---|
shows the purification scheme of S. solfataricus ß-glycosidase (mutant S101C) expressed in Escherichia coli (EcSßgly_S101C) highlighting its similarity to that of the wild-type protein previously expressed, after cloning, in E. coli (Moracci et al. 1995). Sonication on bacterial pulp and a thermoprecipitation step on the homogenate made it possible to purify ß-glycosidase from the major fraction of proteins contained in E. coli. Further hydrophobic and gel filtration chromatography steps were needed to obtain the homogeneous enzyme. Purity was tested by reverse phase HPLC and SDS-PAGE. The specific activity at 75°C of EcSßgly_S101C was 175 u /mg, thus comparable to that of EcSßgly (180 u/mg) (Moracci et al. 1995).
|
) in the proximity of the catalytic tunnel, with the -SH groups located away from the -COOH of the essential nucleophile Glu-387 in the active site, for about 19.67 Å. The Ser-101 is located in a nonstructured and solvent-exposed region of the protein structure. After substitution with a cysteine, if a similar spatial disposition is assumed for the SH group with respect to the OH group of serine maintains, C101 (shown orange in Fig. 1) is distant 38 Å from the catalytic nucleophile Glu-387.
|
) does not indicate a discontinuity point in the slope in the temperature range 298–358 K. The same occurs for mutant protein (data not shown). These results do not suggest any conformational change induced by temperature in the investigated range. The insert of Figure 2 shows the temperature dependence of the catalytic activity of EcSßgly_S101C and EcSßgly in the range 20°–90°C. It would appear that the curves can be superimposed and reversed in the explored range of temperature, thus demonstrating that the substitution does not alter the function of the enzyme.
|
The kinetics of the reaction with the 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) reagent is shown in Figure 3. The reaction time observed in EcSßgly (4 cysteines saturated after 3 h) suggests that the reactivity of these cysteines is very low in agreement with their solvent exposition of 11.9%. However, EcSßgly_S101C, which possesses two free cysteines/subunit, reacted with four molecules of DTNB/tetramer with a biphasic kinetics, involving a first step occurring after 5 min and a slower step occurring after 
3 h. The large difference in the reaction rate of Cys-344 and Cys-101 made it possible to selectively label EcSßgly_S101C at position 101. The specific activity of EcSßgly and EcSßgly_S101C labeled by the spin label is not modified after labeling.
|
shows the temperature dependence of far and near UV CD spectra of EcSßgly in the interval ranging from 20° to 80°C. Only small changes are observed in the CD spectra suggesting that both secondary and tertiary structures of the enzyme are poorly affected by the temperature increase.
|
(spectra a and b, respectively) at temperature of 298 K. Both the spectra consist of two sets of hyperfine lines: three strong components indicated by B-1, B0, and B+1, and less intensive and broad peaks indicated by A1 and A2. The EPR spectra were performed in a temperature range of 20°–70°C. The spectra repeated on protein samples after cooling to room temperature were superimposable, thus indicating the reversibility of the changes observed in the EPR spectra. The parallel hyperfine splitting (A'zz) between the low (A1) and high field (A2) components is the relevant experimental parameter for the dynamic characterization of the spin label bound to the protein macromolecule, which is measured by the rotational correlation time 
c. The rotation correlation times c are calculated from the equation
|
 |
and compared to that of EcSßgly-MAR•. Within the temperature range under investigation, the c = 1/c values vary from 6 x 107 to 2 x 108 sec-1 for EcSßgly-MAR•, and from 5.62 x 107 to 1.1 x 108 sec-1 for EcSßgly_S101C-MAR•. These values are essentially lower than 5 x 1010 sec-1, which is a typical value of frequency rotation for free nitroxides in nonviscous solutions. On the other hand, they are significantly higher than the rotation frequency of the enzyme globule as a whole, which was estimated for a protein with molecular weight of 240,000 Mr as c 
5 x 106 sec-1. Thus, the c value is determined by the mobility of the nitroxide itself on the protein matrix reflecting the local protein dynamics in the vicinity of -SH groups. The Arrhenius plot for EcSßgly-MAR•, which is shown as wide circles in Figure 5B, highlights a discontinuity point at 311 K in the slope, whereas the EcSßgly_S101C-MAR• shows a monotone increase in the motional regime below and above the same temperature. Consequently, it is possible to calculate the enthalpy and entropy related to the spin-label rotation inside the protein matrix activation using the different lines and slopes, on the base of the equation
 |
. For EcSßgly-MAR•, the values of apparent enthalpy activation are 9.88 kJ/mol at temperatures below 311 K and 6.68 kJ/mol at higher temperatures. The corresponding values for the apparent activation entropy are -5.88 e.u. and -11.12 e.u. for temperatures below and above the inflection point, respectively.
|
Frequency domain fluorometry studies of tryptophanylic residues
The tryptophanyl emission decay properties of EcSßgly at neutral pH were investigated by frequency domain fluorometry. The phase shifts and the demodulation factors were collected upon excitation at 295 nm to exclude tyrosine contribution to the whole emission and were analyzed as described in Materials and Methods. The upper part of Figure 6 shows the fluorescence lifetime distribution at 25°C. The tryptophanyl lifetime distribution observed for the ß-glycosidase from S. solfataricus but expressed in E. coli appears to be similar to that reported for the ß-glycosidase wild type (Bismuto et al. 1997). The temperature dependence of lifetime distribution parameters also appears to be superimposable. Table 3 shows the distribution center values of long-lived and short-lived components of EcSßgly at increasing temperatures. An Arrhenius plot of these values is shown in the lower part of Figure 6. Two different slopes are observed for both the lifetime distribution components. The different linear dependences of the center values are analyzed on the basis of the equation:
|
|
 |
reports the activation energies of the different slopes of the centers.
|
|
Discussion |
|---|
|
TOPAbstractIntroductionResultsDiscussionConclusionMaterials and methodsReferences |
|---|
). Moreover, temperature increase does not cause significant structural rearrangements in the secondary and tertiary organization of the protein as observed by circular dichroism measurements either in the far or near UV regions. However, the inability to observe discontinuity points in the temperature dependence of enzymatic activity does not correspond to the absence of changes in the conformational dynamics of the protein structure. A protein solution undergoes constant random thermal motions within a stable equilibrium structure. These motions involve displacements of individual atoms, bonds, functional groups, side chains, local regions of the backbone, secondary structure elements, and entire folded domains. However, the links between the dynamics and catalysis remain unclear. In fact, in a given protein macromolecule, only a subset of motions is important for biological function. The challenge is to identify functionally relevant motions. Effective and detailed approaches to investigate local and global motions that run across the protein structure are obtained by means of modern EPR labeling and time-resolved fluorescence techniques (Likhtenshtein 1976Likhtenshtein 1993; Alcalà et al. 1987). The EPR measurements of rotation correlation times of EcSßgly-MAR• and EcSßgly_S101-MAR•, mainly concern the local dynamics at 344 and 101 residues. A different behavior is observed in the Arrhenius plot of frequency correlation of EPR spin-label rotation that appears to be monotonic linearly dependent on temperature for EcSßgly_S101-MAR•, in contrast with the biphasic linear dependence observed for EcSßgly-MAR•, which shows a discontinuity in the slope at 311 K, indicating different local dynamics of the protein at different sites. The change in the slope above 311 K suggests a transition to a different local spatial arrangement of thermophilic ß-glycosidase and, because of this, a decrease in flexibility occurs. This conclusion is corroborated by the activation parameters 
H
and S obtained from the two different regions of the Arrhenius plot. In fact, the observed values of apparent enthalpy and entropy activation of rotation were found to be smaller at T > Tin than those for the low temperature region (Table 2). This means that the protein structure becomes more rigid as suggested by the values of model compounds (Table 2) and in agreement with the known and accepted models for the thermostable proteins that are intrinsically rigid polymers as a necessary condition for their thermostability (Fontana 1988). However, the Arrhenius plot relative to residue 101 shows intermediate values (7.45 kJ/mol for the apparent activation enthalpy and -10.09 u.e for entropy, respectively) suggesting that the local flexibility/rigidity as seen at position 101 is comparable to the conformational mobility observed at position 344 at temperatures higher than 311 K.
The global structural dynamics of EcSßgly can be studied by examining the tryptophanyl emission decay by frequency domain fluorometry (Beechem and Brand 1985; Alcalà et al. 1987; Nishimoto et al. 1998). The sensitivity of tryptophanyl fluorescence lifetime to a wide variety of microenvironmental conditions has been well documented. Solvent molecules and intramolecular groups likely to interact with tryptophan residues can quench the fluorescence and nonradiative energy transfer processes also can occur (Chen and Barkley 1998). The dynamic quenching processes that decrease the fluorescence lifetime are determined by structural fluctuations in the protein matrix. The emission decay of EcSßgly is heterogeneous and can be interpreted in terms of a bimodal distribution of fluorescence lifetimes (Fig. 4A). The center and width values of the components of the bimodal lifetime distribution of EcSßgly are similar to those obtained for the Sßgly, which were associated with two different classes of tryptophanyl residues: The long-lived distribution component, whose center was at 7.4 nsec, includes tryptophanyl residues located in buried regions with high rigidity (blue in Fig. 1); the short distribution component, whose center was at 2.6 nsec, corresponds to tryptophans embedded in more flexible and exposed regions (red in Fig. 1) (Bismuto et al. 1997, 1999; D’Auria et al. 1997). Molecular dynamic simulation was used to calculate the tryptophanyl lifetime of each of the 17 residues per subunit of EcSßgly and the comparison with the values observed confirms the above reported structural and dynamical conclusions (Bismuto et al. 2000). Table 3 shows the centers of the short- and long-lived distribution component of EcSßgly emission decay as a function of temperature, which also appear to be close to those reported for the Sßgly (Bismuto et al. 1997). The Arrhenius plot of the center values reported in Table 3 are shown in Figure 6B. Both the dependences of long-lived and short-lived components of the lifetime distribution show a discontinuity in the slope at 328 K and 336 K, respectively. Table 4 shows the Arrhenius parameters obtained by linear best-fits regarding data reported in Figure 6B. At temperatures below 328 K, the apparent activation energy of the dynamic processes induced by temperature increase and causing fluorescence quenching is quite similar for both the long-lived and short-lived components of the fluorescence lifetime distribution (i.e., 7.69 and 6.93 kJ/mol, respectively). The same occurs for the activation energies above 336 K, where the values are 23.22 and 18.03 kJ/mol for long-lived and short-lived components, respectively. This observation proves that the changes in the slope indicate conformational changes involving the entire protein matrix, which increases its susceptibility to the thermal insult as a consequence of larger flexibility. Therefore, the conformational changes observed in the temperature range 328–336 K for long- and short-lived distribution components, respectively, produce a decrease in the average of the energetic barriers relative to the collisional processes causing fluorescence quenching.
|
Conclusion |
|---|
|
TOPAbstractIntroductionResultsDiscussionConclusionMaterials and methodsReferences |
|---|
|
Materials and methods |
|---|
|
TOPAbstractIntroductionResultsDiscussionConclusionMaterials and methodsReferences |
|---|
Site-directed mutagenesis
The EcSßgly_S101C was prepared by site-directed mutagenesis following the Mikaelian and Sergeant method (Mikaelian and Sergeant 1992) based on the polymerase chain reaction (PCR). The mutagenic oligonucleotide (Amersham Pharmacia) was as follows (mismatches are underlined): 5'-AAACTTTGATGAATGCAAA CAAGATGTGA-3'.
The amplified product was cloned in a plasmid vector pGEM3 (Promega) so that the gene was under the control of a T7 RNA polymerase promoter. The mutation was identified by direct sequencing and the correctness of the mutagenized gene was confirmed by sequencing the complete gene.
Protein concentration, enzymatic assay, and kinetic constants
Protein concentration was determined by Bradford (1976) and by Lowry et al. (1951) methods, using bovine serum albumin as standard. For EPR, circular dichroism, and fluorescence analyses, the protein concentration was determined at 280 nm using an 
M of 9.5 x 105 cm-1 M-1. The activity of protein samples that were free and modified by the spin label was tested according to the standard procedure described in Nucci et al. (1993). The kinetic constants of free and MAR•-labeled protein samples were measured under standard assay conditions using the substrate (ONPG or PNPG) in the concentration range of 0.2–24 mM, from 30°–70°C. All the protein kinetics were repeated several times and all data were analyzed by the program Grafit 3.0 (Erithacus Software- R.J. Leatherbarrow).
SH group determination in EcSßgly and S101C by Ellman analysis
The number of the free thiols in the EcSßgly and EcSßgly_S101C was performed by DTNB reaction according to Ellman assay (1959). Aliquots of EcSßgly_S101C had been initially subjected to G25 gel filtration chromatography and dyalised against 0.1 M sodium phosphate buffer at pH 7.5 to remove the DTT from the sample. Two and 4 nM of EcSßgly or EcSßgly_S101C samples were transferred into a quartz cuvette (1-cm length path) containing 0.1 M sodium phosphate buffer at pH 7.5 and 0.25 mM DTNB for a final volume of 1 mL. The reaction was followed spectrophotometrically at 25°C by a Varian spectrophotometer Cary 1. After 4 h of incubation, the increase in absorbance at 412 nm due to the release of the 2-nitro-5-thiobenzene anion (412 = 13.6 x 103 M-1 cm-1) was detected against a reference cuvette containing all reactants except the proteins.
Enzyme thermal activities
EcSßgly and EcSßgly_S101C dependence on temperature of enzymatic activity was determined by assaying enzyme aliquots of 4.1 pmoles in a 1-mL sample mixture containing 50 mM sodium phosphate buffer at pH 6.5 and 2.8 mM ONPG as substrate in the temperature range of 25°–90°C. To determine the Arrhenius plot for EcSßgly and EcSßgly_S101C, the data of enzymatic activity were acquired in the above condition every 2.5°C ± 0.1°C.
Far and near UV-CD spectroscopy
Circular dichroism measurements were performed on samples of EcSßgly and EcSßgly_S101C, which were both labeled and not labeled by MAR•, at protein concentrations of 0.41 µM (far-UV) and 1.67 µM (near-UV) in 50 mM sodium phosphate at pH 7.0. A model J-710 spectropolarimeter (Jasco, Tokyo, Japan), which was equipped with a Neslab RTE-110 temperature controller (Neslab Instruments, Portsmouth, NH) and calibrated with a standard solution of (+)-10-camphorsulfonic acid. The cuvettes of 0.1- and 1.0-cm path length (Hellma, Jamaica, NY) were used in the far (190–240 nm) and near UV (250–310 nm) region, respectively. Photomultiplier absorbance did not exceed 600 V in the spectral regions measured. Each spectrum was signal-averaged at least five times, smoothed with Spectropolarimeter System Software Ver. 1.00 (Jasco), and baseline-corrected by subtracting the buffer spectrum. All measurements were performed at the temperatures indicated under a nitrogen flow (3 L/h).
Preparation of samples for EPR measurements
A mixture of 400 µM MAR• and 16.3 µM EcSßgly in a 0.1 M phosphate buffer at pH 6.5 was incubated in a thermostatic bath at 35°C for 96 h until the saturation of the protein by MAR•, monitored by the EPR signal, was achieved. The excess of free MAR• was removed from the solution by gel filtration chromatography (G25 Pharmacia). The sample was concentrated on Centricon 30 (Amicon) at 3,000g and the final concentration of EcSßgly was estimated to be 16.6 µM by UV (Varian spectrophotometer Cary 1) at 280 nm ( = 9.5 x 105 cm-1 M-1).
To prepare samples of EcSßgly_S101C for MAR• labeling, the DTT was removed from aliquots of 4.1 µM of EcSßgly_S101C by gel filtration chromatography on G25 Pharmacia (Amersham Pharmacia Biotech) and the sample was concentrated by Centricon 30 at 3,000g. A mixture of 400 µM MAR• and 15.8 µM of S101C in 0.1 M sodium phosphate buffer at pH 6.5 was incubated in a thermostatic bath at 35°C for 24 h until the saturation of mutated cysteines by MAR• was achieved. The modification process was monitored by electron spin resonance. The excess of free MAR• was removed from the solution by gel filtration chromatography (G25 Pharmacia) and the sample concentrated on Centricon 30 at the final concentration of 17 µM.
The number of MAR• label per one molecule of EcSßgly-MAR• and EcSßgly_S101C-MAR• (e.g., the degree of modification) was calculated as a ratio of moles of bound label on moles of protein by double integration of the EPR spectrum of purified labeled proteins.
EPR spectroscopy
EPR spectra were measured by a Bruker EMX-220 X-band ( = 9.4 GHz) digital EPR spectrometer equipped with Bruker ET 4210 variable temperature accessories. For all measurements, the liquid samples were placed in the narrow (1-mm innerdiameter) end of glass Pasteur pipettes, with no EPR signal within the region of interest (g = 2.00). The sealed capillary tube was filled to >20 mm in length (100 µL) and centered in the rectangular TE102 cavity. To prevent samples overheating during the measurement of temperature dependencies, the stabilization of the temperature at each point was performed with the sample obtained from the cavity. The sample was then returned and kept at least 10 min for temperature equilibrium purposes before the recording was taken. The deviation from the setting point during the measurements did not exceed 0.1 K.
All spectra were recorded at a nonsaturating microwave power of 20 mW, 100 kHz magnetic field modulation of 2 G amplitude, and receiver gains in a range from 2 x 104 to 1 x 105. Digital field resolution was 1,024 points per spectrum, allowing all hyperfine splittings to be measured directly with an accuracy >0.1 G. Spectra processing (spectral algebra, digital filtering, differentiation, numerical integration, and so on) and measurements of the splitting constants were performed using Bruker WIN-EPR software.
Fluorescence -emission decay measurements
Frequency-domain techniques were used to measure the fluorescence decay of the EcSßgly sample in the range 5–200 Mhz using a multifrequency phase shift and modulation cross-correlation fluorometer GREG 200 (ISS). The emission was observed using an optical filter combination of UV 34 and U340 (Oriel Corp.) and the reference was a glycogen solution used as a scatterer. The temperature was monitored continuously during measurements by attaching a thermocouple to the sample cuvette. Readings of the thermocouple were monitored by an Omega Digicator (=mega Engineering) with an accuracy of ±0.1°C. The absorbance of the protein solution did not exceed 0.1 at the excitation wavelength. The lifetime analysis was performed by Global Unlimited (University of Illinois at Urbana) according to Beechem (1992).
Structural analysis
The structural calculation of atomic distances and secondary structure assignment on the ß-glycosidase three-dimensional structure (PDB code 1GOW) were performed by the software Swiss-PdbViewer ver. 3.7 (Guex and Peitsch 1997), whereas the solvent-exposed surface was calculated by the software Molmol ver 2k.1 (Koradi et al. 1996) using a standard radius of 1.4 Å.
|
Acknowledgments |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
References |
|---|
|
TOPAbstractIntroductionResultsDiscussionConclusionMaterials and methodsReferences |
|---|
Alcalà, R., Gratton, E., and Prendergast, F. 1987. Fluorescence life-time distribution in proteins. Biophys. J. 51: 597–604.
Beechem, J. 1992. Global analysis of biochemical and biophysical data. Methods Enzymol. 210: 37–54.[Medline]
Beechem, J. and Brand, L. 1985. Time resolved fluorescence decay in proteins. Annu. Rev. Biochem. 54: 43–71.[CrossRef][Medline]
Bismuto, E., Irace, G., D’Auria, S., Rossi, M., and Nucci, R. 1997. Multitryptophan-fluorescence decay of ß-glycosidase from the extremely thermophilic archaeon Sulfolobus solfataricus. Eur. J. Biochem. 244: 53–58.[Medline]
Bismuto, E., Nucci, R., Rossi, M., and Irace, G. 1999. Structural and dynamic aspects of ß-glycosidase from mesophilic and thermophilic bacteria by multitryptophanyl emission decay studies. Proteins 2: 35–45.
Bismuto, E., Martelli, P.L., Casadio, R., and Irace, G. 2000. Tryptophanyl fluorescence lifetime distribution of hyperthermophilic ß-glycosidase from molecular dynamics simulation: A comparison with the experimental data. Prot. Sci. 9: 1730–1742.[Abstract]
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.[CrossRef][Medline]
Carugo, O. and Argos, P. 1997. TITOLO. Protein Engineering 10: 777–787.
Chen, Y. and Barkley, M. 1998. Toward understanding tryptophan fluorescence in proteins. Biochemistry 37: 9976–9982.[CrossRef][Medline]
D’Auria, S., Rossi, M., Nucci, R., Irace, G., and Bismuto, E. 1997. Perturbation of conformational dynamics, enzymatic activity, and thermostability of ß-glycosidase from Archaeon Sulfolobus solfataricus by pH and sodium dodecyl sulphate detergent. Proteins 27: 71–79.[CrossRef][Medline]
Ellman, G.L. 1959. Tissue sulfydryl groups. Arch. Biochem. Biophys. 82: 70–77.[CrossRef][Medline]
Freed, J.H. 1976. Theory of slow tumbling ESR spectra for nitroxides. In: Spin Labelling. Theory and Applications (ed. L.J. Berliner), pp. 64–155. Academic Press, New York, NY.
Fontana, A. 1988. Structure and stability of thermophilic enzymes. Biophys. Chem. 29: 181–183.[CrossRef][Medline]
Gratton, E. and Limkeman, M. 1983. A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys. J. 44: 315–324.
Guex, N. and Peitsch, M.C. 1997. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714–2723.[CrossRef][Medline]
Hober, B., Jurgens, C., Wilmanns, M., and Sterner, R. 2001. Stability, catalytic versatility and evolution of the (ß
)8 barrel fold. Curr. Opin. Biotechnol. 12: 376–381.[CrossRef][Medline]
Koradi, R., Billeter, M., and Wüthrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structures J. Mol. Graphics 14: 51–55.[CrossRef][Medline]
Likhtenshtein, G.I. 1976. Water and protein dynamics. In Spin labelling: Theory and application, Vol. 2 (ed. L.J. Berliner), pp. 45–53. Academic Press, New York, NY.
———. 1993. Biophysical labelling method in molecular biology. Cambridge University Press, New York, NY.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275.
Mikaelian, I. and Sergeant, A. 1992. A general and fast method to generate multiple site directed mutations. Nucleic Acid Res. 20: 376.
Moracci, M., Nucci, R., Febbraio, F., Vaccaro, C., Vespa, N., La Cara, F., and Rossi, M. 1995. Expression and extensive characterization of a ß-glycosidase from the extreme thermoacidophilic archaeon Sulfolobus solfataricus in Escherichia coli. Enzyme Microb. Technol. 17: 992–997.
Nishimoto, E., Yamashita, S., Szabo, A.G., and Imoto, T. 1998. Internal motion of lysozime studied by time-resolved fluorescence depolarization of tryptophan residues. Biochemistry 37: 5599–5607.[CrossRef][Medline]
Nucci, R., Moracci, M., Vaccaro, C., Vespa, N., and Rossi, M. 1993. Exo-glucosidase activity and substrate specificity of the ß-glycosidase from Sulfolobus solfataricus. Biotechnol. Appl. Bioch. 17: 239–250.
Pearl, L.H, Hemmings, A.M., Nucci, R., and Rossi, M. 1993. Crystallisation and preliminary X-ray analysis of the ß-galactosidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. J. Mol. Biol. 229: 561–563.[CrossRef][Medline]
Zavodsky, P., Kardos, J., Svingor, A., and Petsko, G.A. 1998. Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc. Natl. Acad. Sci. 95: 7406–7411.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
