Adenylate Kinase fromSulfolobus acidocaldarius:Expression inEscherichia coliand Characterization by Fourier Transform Infrared Spectroscopy

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 333, No. 1, September 1, pp. 75–84, 1996 Article No. 0366

Adenylate Kinase from Sulfolobus acidocaldarius: Expression in Escherichia coli and Characterization by Fourier Transform Infrared Spectroscopy Heiko Bo¨nisch,* Jan Backmann,†,1 Thomas Kath,* Dieter Naumann,† and Gu¨nter Scha¨fer*,2 *Institute of Biochemistry, Medical University of Lu¨beck, Ratzeburger Allee 160, 23538 Lu¨beck, Germany; and †Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany

Received May 20, 1996

Adenylate kinase from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius has been overexpressed in Escherichia coli. The highly purified enzyme was characterized by Fourier transform infrared spectroscopy (FTIR). Analysis of FTIR spectra and estimation of secondary structure revealed a global protein structure similar to that of other adenylate kinases. Thermal unfolding of the protein with an estimated Tm value near 907C is irreversible due to protein aggregation. The enzyme exhibits long-term stability up to 807C, which is an excellent adaptation to the physiological growth temperature of 75–807C. Halfwidths of secondary-structure-sensitive bands and hydrogen–deuterium exchange experiments revealed that in comparison to adenylate kinase from porcine muscle cytosol the Sulfolobus enzyme is characterized by a significantly more compact and rigid protein core structure, which is likely to contribute specifically to the extreme thermostability of the protein. q 1996 Academic Press, Inc.

Key Words: adenylate kinase; FTIR spectroscopy; Sulfolobus acidocaldarius; archaea; protein structure.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is involved in the reversible transfer of terminal phosphate groups between the adenine nucleotides (ATP / AMP S 2 ADP), a reaction obviously indispensible for the energy metabolismn of procaryotic and eucaryotic cells. Accordingly, adenylate kinases have been found to be present in all organisms and 1 Present address: Vrije Universiteit Brussel, Instituut voor Moleculaire Biologie, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium. 2 To whom correspondence should be addressed. Fax: //49-4515004060.

tissues investigated to date. For some adenylate kinases the three-dimensional structure has been solved by X-ray crystallography (1–5). The adenylate kinase from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius, growing optimally at 75–807C and pH 2–3 (6), was the first example of an archaebacterial adenylate kinase purified to homogeneity and characterized enzymatically (7). Identification of the adk gene, cloning, and overexpression of the protein in Escherichia coli (8) allow a large-scale preparation of highly purified enzyme, the prerequisite for detailed biophysical investigations, as described in this paper. Due to the very low homology in primary structure compared to all other known adenylate kinases and the extreme thermostability of this enzyme (7, 8), it is of particular interest to gain information about the structure of this adenylate kinase. In this work Fourier transform infrared (FTIR)3 spectroscopy was used to compare adenylate kinase from S. acidocaldarius to the cytosolic adenylate kinases from porcine and rabbit muscle. We expected a first hint as to whether the unusual primary structure of the Sulfolobus enzyme is correlated to an altered three-dimensional structure. Further, this technique was applied to monitoring the temperature-induced changes in molecular structure and dynamics, first, to characterize the temperature behavior of the protein and, second, to search for possible relations between structure and thermostability. MATERIALS AND METHODS Materials. Cytosolic adenylate kinase (AK1) from porcine muscle and rabbit muscle was purchased from Boehringer Mannheim GmbH 3 Abbreviations used: FTIR, Fourier transform infrared; H/D exchange, hydrogen–deuterium exchange; IPTG, isopropyl-1-thio-b-Dgalactopyranoside; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Ap5A, diadenosine-5,5*-pentaphosphate.

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(Germany). Cellulose phosphate cation exchanger (medium mesh) was purchased from Sigma Chemie GmbH (Deisenhofen, Germany); Blue Sepharose Cl-6B was from Pharmacia Biotech (Uppsala, Sweden). All chemicals were obtained from commercial sources at the highest purity available. Overproduction of S. acidocaldarius adk in E. coli. Limited expression of the S. acidocaldarius adk gene when left under the control of the inducible T7 lac promoter in vector pET11a (Novagen) has already been demonstrated (8). To increase expression the adk coding region was cut with NdeI and BamHI from pET11a and ligated into vector pET3a (Novagen). In the resulting plasmid the adk gene was under the control of a ‘‘plain’’ T7 promoter. The plasmid was used to transform E. coli strain Bl 21 (DE3). To obtain the optimal strain for expression, individual clones were grown until the OD600 reached 0.6 and induced for 3 h by addition of 0.1–0.5 mM IPTG. Expression was checked by analysis of the total cell protein subjected to SDS–PAGE (9) and by determination of the specific activity of adenylate kinase at 907C in the cytosolic extract. The correct in-frame cloning was verified by sequencing. The chosen strain showed strong overexpression on addition of 0.1 mM IPTG, but under the applied growth conditions (below) a strong basal expression was also observed. Preparation of recombinant S. acidocaldarius adenylate kinase from E. coli. E. coli strains were grown at 377C in yeast–peptone medium with 50 mg/ml ampicillin, induced after 3 h with 0.1 mM IPTG. In the stationary phase, cells were harvested by centrifugation, resuspended in 1 mM EDTA, 20 mM Tris–HCl, pH 6.0, and stored at 0807C. For each preparation about 120g of cells (wet weight; from a 20-liter culture volume) was thawed and disrupted by ultrasonication (5 1 30 s, 150 W, 20,000 Hz); cell debris was removed by centrifugation. The cytosolic extract was brought carefully to pH 2.0 with 2 N HCl, slowly adjusted to pH 6.0 with 2 N NaOH, and stirred for at least 30 min at 47C. Afterward, denatured protein was removed by centrifugation. The supernatant was applied to a cellulose phosphate column (5 1 20 cm, 400 ml; 1 mM EDTA, 20 mM Tris–HCl, pH 6.0; 3 ml/min) and bound proteins were eluted with a linear gradient from 0 to 1000 mM NaCl. Activity-containing fractions were pooled and concentrated by ultrafiltration on an Amicon PM-10 membrane. After adjustment to pH 7.5 and 600 mM NaCl, the eluent was loaded on a Blue Sepharose column (2.5 1 15.2 cm, 75 ml; 1 mM EDTA, 900 mM NaCl, 20 mM Tris–HCl, pH 7.5; 3 ml/ min). The column was washed with 1 mM EDTA, 900 mM NaCl, 20 mM Tris–HCl, pH 7.5, and 1 mM EDTA, 200 mM NaCl, 20 mM Tris– HCl, pH 7.5. Adenylate kinase was eluted with 100 ml each of 4 mM ATP, AMP, MgCl2 in 1 mM EDTA, 200 mM NaCl, 20 mM Tris–HCl, pH 7.5, and 10 mM ATP, AMP, MgCl2 in 1 mM EDTA, 200 mM NaCl, 20 mM Tris–HCl, pH 6.0. The fractions of both eluent peaks were combined. The protein was further purified by gel filtration, first, on a Superdex 75 column (Pharmacia HiLoad, 16/60, prep grade), followed by two runs on a Bio-Rad Biosil SEC 250 (5 mM EDTA, 100 mM Tris–HCl, pH 7.5, 1 ml/min). Protein-containing fractions were pooled, dialyzed against double-distilled water, concentrated by ultrafiltration, and lyophilized for storage. The purity of the preparation was checked by SDS–PAGE (9) with silver staining (10). Protein determination was performed according to Peterson (11). Purification of cytosolic adenylate kinase from porcine and rabbit muscle. For FTIR-spectroscopic measurements the commercial preparations of adenylate kinase from porcine and rabbit muscle were subjected to further purification. The ammonium sulfate suspensions were dissolved in 100 ml 200 mM NaCl, 50 mM Tris–HCl, pH 7.0, and loaded on a Blue Sepharose column (100 ml, 1 ml/min) equilibrated with buffer. Adenylate kinase was eluted with 100 ml 0.1 mM Ap5A. Separation from bound Ap5A, buffer exchange to 100 mM sodium cacodylate, pH 7.0 (H2O), and concentration were accomplished by ultrafiltration. Purity and homogeneity of the proteins were checked by SDS–PAGE (9) with silver staining (10) and gel filtration (Bio-Rad Biosil SEC 250; 5 mM EDTA, 100 mM Tris–HCl,

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pH 7.5; 1 ml/min). For H/D-exchange experiments purified adenylate kinase from porcine muscle was lyophilized in 100 mM sodium cacodylate, pH 7.0 (H2O). Enzyme assay. During the preparation, the enzyme activity of adenylate kinase from S. acidocaldarius was determined at 907C in the direction of ADP formation according to Lacher and Scha¨fer (7). The enzyme activity of adenylate kinase from porcine and rabbit muscle cytosol was measured according to Brolin (12). Enzyme stability. For investigation of the long-term thermostability of adenylate kinase from S. acidocaldarius, 50 mg/ml protein was dissolved in 50 mM sodium cacodylate buffer, pH 6.0. In screwcap reaction vessels, each of five 2-ml portions was incubated in a Haake N3 water bath at 70, 80, 85, and 907C for 10 h. Every hour 100-ml samples were withdrawn and activity was tested by the coupled enzyme assay at 457C (7). FTIR-spectroscopic measurements. Lyophilized adenylate kinase from S. acidocaldarius was dissolved to a concentration of approx 20 mg/ml in 100 mM sodium cacodylate, pH 7.0 (H2O). Adenylate kinase from porcine and rabbit muscle cytosol in 100 mM sodium cacodylate, pH 7.0 (H2O), was concentrated to approx 20 mg/ml. The solutions were centrifuged at 8000g at 57C for 10 min to precipitate possible impurities. Infrared spectra were recorded using a Bruker IFS-28/B FTIR spectrometer equipped with a DTGS detector. The protein solutions were placed in a thermostated cuvette equipped with CaF2 windows. The pathlength was 8 mm. To compensate for H2O absorption, the buffer solutions were placed in the same cell but with slightly (1–2%) shorter path lengths. FTIR spectra of the protein and the buffer solutions were recorded at a nominal resolution of 4 cm01. The spectra were Fourier deconvoluted using the OPUS2.0 software of Bruker Analytische Messtechnik (Karlsruhe, Germany). A Lorentz lineshape function, a deconvolution factor (maximal value of the multiplied amplifying function at the end of the interferogram) of 4000, and a noise reduction factor (the fraction of the interferogram at which the Blackman apodization function subsides to zero) of 0.3 were applied. The deconvoluted spectra were fitted with Gaussian band profiles using the Levenberg–Marquart procedure and applying the Bruker OPUS2.0 software. The number and position of the peaks were taken from the derivative and the deconvoluted spectra. Temperature gradient studies. Lyophilized adenylate kinase from S. acidocaldarius and porcine muscle cytosol were dissolved to a concentration of approx 20 mg/ml in 100 mM sodium cacodylate, pH 7.0 (S. acidocaldarius, H2O and D2O; porcine muscle, D2O). The solutions were centrifuged 10 min at 8000g at 57C to precipitate possible impurities. The protein solutions were placed in a homemade, demountable cell equipped with CaF2 windows. The path length was 6 mm (H2O) or 50 mm (D2O). To compensate for H2O/D2O absorption, the buffer solutions were placed in the same cell but with slightly (1–2%) shorter path lengths. The protein solutions were equilibrated for 60 min at 207C; afterward the temperature gradient was started. The temperature of the gas-tight IR cell device was controlled by a programmable circulator purchased from Haake, Germany. Infrared spectra were recorded using a Bruker IFS-66 FTIR spectrometer equipped with a water-cooled globar and a DTGS detector as described recently (13). FTIR spectra of the protein and the buffer solutions were recorded at a nominal resolution of 4 cm01. While the temperature of the sample was linearly increased at a rate of 0.57/min, 227 interferograms/17 temperature change were collected, averaged, and Fourier transformed using a Boxcar apodization function. The spectra were Fourier deconvoluted using the OPUS2.0 software of Bruker Analytische Messtechnik (Karlsruhe, Germany). A Lorentz lineshape function, a deconvolution factor of 2000, and a noise reduction factor of 0.5 were applied. The plots of intensity vs temperature for selected infrared marker bands were evaluated from the original or deconvoluted spectra. Isothermal studies. The spectroscopic equipment and the sample preparation (using D2O buffer) were the same as for the temperature

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ADENYLATE KINASE FROM Sulfolobus acidocaldarius TABLE I

Protocol for Purification of Recombinant S. acidocaldarius Adenylate Kinase from E. coli

Purification step

Protein (mg)

Activity (U)

Sp act (U/mg)

Yield (%)

Enrichment

Supernatant ultrasound treatment Supernatant acid treatment Eluent cellulose phosphate Eluent Blue Sepharose Eluent gel filtration

10,500 2,940 400 78 40

2,050,000 1,290,000 164,000 68,600 46,300

195 439 410 880 1157

100 63 8 3.3 2.3

1.0 2.2 2.1 4.5 6.0

Note. A typical example is shown. The activity data refer to 907C. All fractions were assayed at this temperature during purification. Cytosolic extract and supernatant from acid treatment contain background activity from E. coli adenylate kinase.

gradient experiments. During an experiment the sample was heated as fast as possible (within approx 5 min from room temperature to the desired temperatures of 70 and 857C, respectively) and kept for several hours at this temperature. The moment of reaching the respective temperature was taken as zero time. The temperature of the gas-tight IR cell device was controlled by a programmable circulating temperature bath purchased from Haake, Germany. The fast temperature enhancement was triggered by a valve directing the stream of circulating liquid into the cell housing. FTIR spectra of the protein and the buffer solutions were recorded at a nominal resolution of 4 cm01. The spectra were Fourier deconvoluted and processed as described in the section on temperature gradient studies.

RESULTS AND DISCUSSION

Enzyme purification. The purification protocol presented above provides the possibility for preparing adenylate kinase from S. acidocaldarius in high purity and quantity, sufficient for detailed biophysical investigations (Table I). This procedure is based on the strong overexpression of the enzyme in the cytosol of E. coli. Specific activity is a factor of 2000 higher than that in the cytosolic extract of S. acidocaldarius. The first purification step, an acid treatment, exploits the unusual acid stability of the enzyme to remove approx 70% of the total protein together with a twofold enrichment of activity. Subsequent chromatography on cellulose phosphate causes large losses of enzyme activity due to incomplete binding, but removes completely the nucleotide binding proteins from E. coli, especially the adenylate kinase, together with approx 85% of the residual protein. The critical purification step for purity is affinity chromatography on Blue Sepharose with specific elution by substrate, yielding an enzyme free of contaminants. Finally, gel filtration serves to obtain a homogeneous preparation of dimeric enzyme free of bound nucleotides and higher aggregates. The yield of this purification procedure on average was 30–40 mg of pure enzyme with a specific activity of 1000 U/mg. Control of purity by SDS–PAGE (Fig. 1) shows a homogeneous single band. The identity of the recombinant protein with the enzyme from S. acidocaldarius was verified by immunoblotting and N-terminal protein sequencing (not shown).

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Resolution-enhanced FTIR spectra of adenylate kinases. Figures 2A–2C show the Fourier self-deconvoluted spectra in the amide I region of adenylate kinase from S. acidocaldarius, porcine, and rabbit muscle cytosol in H2O, respectively. These spectra have been analyzed with the aid of a band-fitting routine; the resulting component bands are depicted in the same figures. Positions and fractional areas of the component bands, including the corresponding literature data for adenylate kinase from E. coli (14), are summarized in Table II. Direct comparison of these spectra reveals an essentially concurrent pattern of component bands for adenylate kinase from porcine and rabbit muscle cytosol, consisting of six bands at 1629–1635, 1640–1643, 1652–1653, 1663–1665, 1673–1678, and 1685–1686 cm01. This spectroscopic similarity coincides well with the assumption of a similar secondary structure of these enzymes anticipated from their high degree of sequence homology. Compared to these mammalian AK1 isoenzymes, adenylate kinase from E. coli con-

FIG. 1. Purification of recombinant S. acidocaldarius adenylate kinase from E. coli. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of fractions from subsequent purification steps. A silverstained gel is shown loaded as follows: lane 1, molecular weight marker; lane 2, 15 mg cytosolic extract; lane 3, 8 mg supernatant acid treatment; lane 4, 4 mg eluent cellulose phosphate chromatography; lane 5, 4 mg eluent from Blue Sepharose; lane 6, 1 mg purified AK after gelfiltration.

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FIG. 2. Fourier self-deconvoluted amide I band contours of adenylate kinase from S. acidocaldarius (A), porcine muscle cytosol (B), and rabbit muscle cytosol (C) in H2O. The spectra have been analyzed with the aid of a band-fitting routine (cf. Materials and Methods); the resulting component bands are depicted in the same figures.

tains an additional insertion of 20 amino acids at the periphery of the molecule, whereas the global folding of the polypeptide chain is comparable, as known from X-ray crystallography (5). Correspondingly, the FTIR spectrum is altered by the occurrence of an additional band at 1648 cm01. Apart from this peculiarity, it shows the characteristic pattern of six component bands at similar wavenumbers (Table II). In comparison to these enzymes, the FTIR spectrum of adenylate kinase from S. acidocaldarius shows apparent similarities with respect to the number and position of amide I component bands. This can be interpreted as evidence for a global protein structure of adenylate kinase from S. acidocaldarius similar to that of adenylate kinase from E. coli, porcine, and rabbit muscle. On the other hand, the FTIR spectrum of Sulfolobus adenylate kinase exhibits significantly reduced bandwidths for all component bands, in particular for the most prominent band at 1654 cm01. The reduced bandwidths evidence low flex-

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ibility and dense packing of the various secondary structure elements, which in turn may contribute to the known high stability of the protein. Hence, although a similar secondary structure is suggested from these data, local deviations in protein structure must be expected. In addition to these more qualitative considerations, the FTIR spectra allow a quantitative estimation of the secondary structure of the adenylate kinases investigated. Using band-narrowing and curve-fitting analysis, this requires the assignment of amide I component bands to different types of secondary structure, based on theoretical considerations from literature and spectra–structure correlations established experimentally (15–22). For a critical discussion of this method see (23). Our assignment of amide I component bands to different types of secondary structure is given in Table II. From these assignments the secondary structure of the adenylate kinases can be calculated as summarized

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ADENYLATE KINASE FROM Sulfolobus acidocaldarius TABLE II

Positions of Amide I Component Bands, Relative Integrated Intensities, and Secondary Structure Assignments for Adenylate Kinase from S. acidocaldarius, Porcine Muscle Cytosol, and Rabbit Muscle Cytosol S. acidocaldarius

Porcine muscle

Rabbit muscle

Band position (cm01)

Band area (%)

Band position (cm01)

Band area (%)

1624 1634 1645

6 13 12

1629

4

1640

20

1654 1663 1670 1678 1687

46 4 7 7 5

1652 1665

46 21

1678 1686

5 4

E. coli (14)

Band position (cm01)

Band area (%)

1635 1643

14 15

1653 1663 1673

39 8 17

1685

5

Band position (cm01)

Band area (%)

1627 1638

5 20

1648 1656

16 43

1671 1681 1688

7 8 1

Assignment

b-sheet b-sheet b-sheet a-helical a-helical, unordered a-helical b-turns b-sheet b-sheet

Note. The results are compared with corresponding data for E. coli measured by Arrondo et al. (14).

in Table III. For the adenylate kinases investigated, the FTIR-spectroscopic analysis reveals different secondary structural compositions: The contribution of ahelical and unordered structures varies from 47 to 67%, the contribution of b-sheet and b-turn structures from 33 to 51%. In this context it must be mentioned that the fraction of b-structures in proteins is generally estimated more accurately by FTIR spectroscopy than the fraction of a-helical structures in contrast to CD spectroscopy. For adenylate kinase from E. coli and porcine

muscle, these data coincide excellently with structural data from X-ray crystallography (Table III), providing evidence for the potential accuracy of this method. Consequently, distinct differences in secondary structure must be expected. Nevertheless, the secondary structure obtained for adenylate kinase from Sulfolobus fits satisfactorily into this group of protein secondary structures. Both the qualitative and quantitative conclusions obtainable from the FTIR analysis lend support to the assumption of similar global protein structures

TABLE III

Comparison of Secondary Structural Data from X-Ray Crystallography and FTIR and CD Spectroscopy for Adenylate Kinase from S. acidocaldarius, E. coli, and Rabbit and Porcine Muscle Cytosol

Enzyme

FTIR

AK S. acidocaldarius

50% a / u 43% b / t 7% t

AK 1 porcine muscle

67% a / u 33% b / t

AK 1 rabbit muscle

47% a / u 34% b / t 17% t

AK E. coli

59% a / u 26% b

X-ray crystallography

61.85% 14.43% 3.09% 11.85% 8.76%

a b 310 t u (2)

50.46% 20.56% 5.14% 12.14% 11.68%

(14)

a b 310 t u (5)

Note. a, a-helical; 310 , 310-helical; b, b-sheet; u, unordered; t, turns.

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46% a 22% b

(24) 40% a / u 50% b 6% t(25)

15% t

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(26)

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FIG. 3. Fourier self-deconvoluted FTIR spectra of adenylate kinase from S. acidocaldarius in the amide I and amide II regions as a function of temperature. The spectra of S. acidocaldarius adenylate kinase in D2O were measured along a temperature gradient between 20 and 1007C.

for adenylate kinase from S. acidocaldarius, E. coli, and porcine and rabbit muscle cytosol. In particular, no indication can be found for the presence of fundamental differences in secondary structure, justifying the assumption of a new class of archaebacterial adenylate kinases. Temperature gradient experiments. Figure 3 shows the spectral changes of adenylate kinase from S. acidocaldarius during a temperature gradient experiment (207C r 1007C r 207C) performed in D2O buffer. Variations in positions and intensities of IR bands in the amide I and amide II regions can be observed as a consequence of temperature-induced alterations in structure and simultaneous H/D exchange. For detailed analysis the peak intensities at 1652 cm01 (pri-

marily a-helical and unordered structures), 1634 cm01 (b-sheet), and 1618 cm01 (b-aggregation band (27)) have been plotted as a function of temperature (Figs. 4A and 4B). From the intensity/temperature course at 1652 and 1634 cm01, it can be seen that thermal denaturation of the protein starts at approx 857C. The apparent Tm value is near 1007C. With decreasing temperature no alteration or restoration of intensity can be observed (Fig. 3); thus, reorganization of secondary structure does not take place. Obviously, thermal unfolding of Sulfolobus adenylate kinase is irreversible. This irreversibility is a consequence of simultaneous aggregation of the protein, as can be seen from the fast increase of intensity of the b-aggregation band at 1618 cm01 above 857C. Due to this irreversible protein aggregation, it is not possible to perform this temperature gradient studies in D2O with protein totally exchanged by previous thermal unfolding. In order to estimate the influence of H/D exchange on the spectral analysis of protein denaturation, the temperature gradient experiments were also performed in H2O, although the signalto-noise ratio of the spectra is considerably lower due to the strong IR absorbance of H2O (not shown). However, the results obtained are comparable except for a shift of the transition curves to lower temperature. The Tm value is near 907C. Due to the coupling of thermal unfolding and irreversible aggregation, thermodynamic equilibrium is not reached. Therefore, the denaturation process is rate-limited; shape and position of the transition curves are dependent on the temperature gradient applied. Exact thermodynamical parameters such as Tm values or van’t Hoff enthalpy of unfolding are not available from these experiments. H/D exchange. Characterization of H/D-exchange processes of peptide backbone amide protons is a pow-

FIG. 4. Intensity–temperature plots for S. acidocaldarius adenylate kinase obtained from the temperature gradient experiment. Peak intensities of three different marker bands as a function of temperature (20–1007C): (A) at 1652 cm01 (a-helices and unordered) and at 1634 cm01 (b-pleated sheet) and (B) at 1618 cm01 (b-aggregates).

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ADENYLATE KINASE FROM Sulfolobus acidocaldarius

FIG. 5. Temperature dependence of the infrared spectra of adenylate kinase from S. acidocaldarius (A) and porcine muscle cytosol (B) in the amide I, II, and II* regions. The spectra of partially H/Dexchanged proteins were measured in D2O along a temperature gradient between 20 and 1007C or 20 and 807C, respectively (ds, down scan). All spectra are shown after band-narrowing by Fourier selfdeconvolution.

erful tool for studying structure, stability, and dynamics of proteins (28, and references therein). This technique is based on the different exchange rates of individual amide protons as a function of the stability of intramolecular hydrogen bonds. Amide protons can be exchanged only if the hydrogen bonds they are involved in are broken (29). Amide protons in the core structures of proteins are found to exchange at extremely low rates. The amount of these slowly exchanging protons is a characteristic of a given protein. This amount can be used to estimate the flexibility and compactness of a native protein. Figures 5A and 5B show the superimposed FTIR spectra of adenylate kinase from S. acidocaldarius and porcine muscle cytosol obtained during a temperature gradient experiment (207C r 1007C or 207C r 807C, respectively) performed in D2O buffer. From the FTIR spectra recorded in H2O (data not shown), in D2O after 1 h incubation at 207C (partially exchanged), and in D2O at 207C after the sample is heated to 100 or 807C, respectively, and cooling back

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to 207C (completely exchanged), the fraction of protons which have not been exchanged after 1 h incubation at 207C can be calculated from the ratio of amide II to amide I integral band intensities. By definition these protons represent the slowly exchanging or persistent protons of these proteins (28). In accordance with this definition 47% of the amide protons of S. acidocaldarius adenylate kinase are slowly exchanging protons, whereas for adenylate kinase from porcine muscle cytosol a fraction of persistent protons of only 13% was calculated. Thus adenylate kinase from Sulfolobus is characterized by a considerably higher fraction of amide protons involved in stable hydrogen bonds, reflecting a definitely more rigid and more densely packed protein structure at 207C in comparison to the enzyme from porcine muscle cytosol. The persisting protons can exchange only if the hydrogen bonds that they are involved in are disrupted by the unfolding of the corresponding protein structure, initiated either by chemical denaturants or by increasing thermal energy. The course of temperature-induced H/D exchange of persisting protons can be followed directly by analyzing the temperature-dependent spectral changes in the H/D-exchange-sensitive amide II region (28): The fractions of unexchanged protons calculated from the residual band intensities at 1546 and 1542 cm01 (normalized using the corresponding amide I intensities) for adenylate kinase from Sulfolobus and porcine muscle were plotted as a function of temperature as shown in Fig. 6. The temperature-induced H/D exchange of persisting protons is a rate-limited process and is consequently dependent on the rate of temperature increase. Further, in the transition region of the protein, the spectral changes observed in both the amide I and the amide II region are due to H/D exchange

FIG. 6. H/D exchange of adenylate kinase from S. acidocaldarius and porcine muscle cytosol during a temperature gradient experiment. Fractions of unexchanged amide protons as a function of temperature were calculated from the normalized amide II intensities at 1546 and 1542 cm01, respectively.

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and the simultaneously occurring protein unfolding processes. However, under the same experimental conditions, these temperature-gradient-induced H/D-exchange curves are characteristic of a given protein. The starting level, the shape of the exchange curves, the temperature TH/D at which the H/D exchange is completed, and the Tm values can be used to compare the flexibility and stability of related proteins (28). From the course of the temperature-induced H/D-exchange curves (Fig. 6), it can be seen that in addition to the considerably higher starting level of unexchanged amide protons, the exchange of persisting protons of S. acidocaldarius adenylate kinase is taking place at significantly higher temperatures compared to adenylate kinase from porcine muscle. TH/D is at 567C for adenylate kinase from porcine muscle and at 977C for Sulfolobus adenylate kinase. Obviously, temperature values around 80–907C are necessary for adenylate kinase from Sulfolobus to attain a flexibility comparable to that of catalytically active mesophilic adenylate kinase from porcine muscle. These results agree well with observations made during comparisons of other homologous proteins from mesophilic and thermophilic organisms, respectively (30). Thermophilic enzymes have evolved to exhibit optimal molecular flexibility for catalytical function at their physiological temperature, hence showing significantly higher rigidity at room temperature in comparison to homologous mesophilic enzymes. Experimental data available from H/D-exchange experiments suggest a distinct correlation between the stability of a protein and its H/D-exchange characteristics: The lower the melting temperature Tm , the lower the temperature TH/D , which naturally is always lower than Tm (31). However, the differences between Tm and TH/D vary, characterizing structural properties of the protein investigated: The larger the difference between Tm and TH/D , the less the H/D exchange is controlled by the global folding/unfolding equilibrium, and the

FIG. 7. Long-term thermostability of adenylate kinase from S. acidocaldarius. Normalized intensity of the IR band at 1618 cm01 (baggregation) as a function of time at 70 and 857C.

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FIG. 8. Thermal inactivation of S. acidocaldarius adenylate kinase. 50 mg/ml enzyme in 50 mM sodium cacodylate, pH 6.0, was incubated for 10 h at 70, 80, 85, and 907C. Samples were withdrawn every hour and activity was measured by the 457C assay. Curve fitting was performed with exponential functions.

higher the structural flexibility under ambient conditions (28). Using the estimated Tm values of 102 and 637C for adenylate kinase from Sulfolobus and porcine muscle, respectively, the differences between Tm and TH/D are calculated as 5 and 77C, respectively. This similarity suggests that adenylate kinases from Sulfolobus and porcine muscle may possess comparable flexibilities at their physiological temperature. Isothermal studies. For a more detailed analysis of possible correlations between enzyme activity, thermal unfolding, and irreversible aggregation, the long-term thermostability of adenylate kinase from S. acidocaldarius has been investigated. Figure 7 gives the time-dependent change in intensity of the b-aggregation band at 1618 cm01 when the protein was incubated in D2O at 70 and 857C, respectively. Figure 8 demonstrates the time course of enzyme inactivation at 70, 80, 85, and 907C. After incubation for 10 h at 70 or 807C, a decrease in enzymatic activity of at most 15% can be observed. The FTIR spectra indicate only slight loss of secondary structure after being kept at 707C for 3 h (not shown) accompanied by a corresponding slight increase in the intensity of the b-aggregation band at 1618 cm01. At temperatures above 807C, long-term incubation results in a strong continuous decrease in enzymatic activity together with a pronounced loss of secondary structure and rapid increase in irreversible aggregation. With the appearance of significant populations of unfolded protein species above 807C protein aggregation removes protein molecules from the folding/unfolding equilibrium until the protein is completely aggregated. The continuous decrease of enzyme activity indicates a progressive thermal inactivation. The extent of enzyme inactivation is dependent on tempera-

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ture and on incubation time. Consequently, the apparent temperature optimum of enzyme activity is dependent on the incubation time as a function of enzyme kinetics and superimposed kinetics of thermal inactivation. The standard activity assay with an incubation time of 60 s reveals an apparent temperature optimum at 907C and high enzymatic activity up to at least 957C (7). With increasing incubation time this value declines to 807C, where the enzyme exhibits long-term stability. This long-term thermostability at 807C in vitro is perfectly in line with the optimal physiological growth temperature of S. acidocaldarius at 75 – 807C. Zhang et al. investigated the thermal unfolding and aggregation of adenylate kinase from rabbit muscle. For this and numerous other mesophilic enzymes it has been shown that enzyme inactivation precedes conformational changes (32). During the temperature increase, the activity of the enzymes is lost prior to changes in backbone conformation, protein unfolding, and aggregation. This has been interpreted as an increased flexibility of the active site with respect to the molecule as a whole. In contrast, the results obtained for Sulfolobus adenylate kinase suggest a more direct relation between enzyme activity and protein unfolding and aggregation. Possibly this must be seen in context with the high rigidity and compact core structure of this small protein molecule. It will be interesting to investigate whether this behavior is a general feature of small thermophilic proteins and is the structural consequence of adaptation to high temperatures of growth. CONCLUSIONS

Adenylate kinase from S. acidocaldarius is characterized by an extremely low homology in primary structure with respect to all other adenylate kinases (8). This raised the question whether this low homology is correlated with an altered protein structure, possibly representing a new class of archaebacterial adenylate kinases. The results presented in this paper are the first experimental biophysical data on the structure and thermostability of an archaebacterial adenylate kinase. For archaebacterial adenylate kinases purified recently from marine methanogenes by Rusnak et al. (33), apart from Sulfolobus enzyme, the only archaebacterial adenylate kinases purified to homogeneity, corresponding data are not yet available. The FTIR-spectroscopic estimation of secondary structure revealed significant similarities to adenylate kinase from E. coli and porcine and rabbit muscle cytosol. In particular, no indication of fundamental differences, i.e., a novel class of archaebacterial adenylate kinases, could be confirmed by FTIR. Rather, all available data suggest that in struc-

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ture and function this enzyme resembles the mesophilic enzymes of the eucaryotic AK 1 type. Investigation of the extreme thermal stability of this enzyme reveals a Tm value near 907C and long-term thermostability up to 807C, representing an optimal adaptation to the physiological growth temperature of 75–807C. Like many other thermophilic proteins, the Sulfolobus enzyme exhibits irreversible thermal unfolding due to irreversible protein aggregation. With regard to the structural basis of thermostability, an increased compactness and rigidity of the protein molecule at room temperature in comparison to proteins from mesophilic sources can be deduced from the experiments performed. ACKNOWLEDGMENTS The technical assistance of Walter Verheyen is gratefully acknowledged. Protein sequencing was performed by Dr. Roland Schmid, Osnabru¨ ck. These investigations have been supported by the Deutsche Forschungsgemeinschaft, Grant Scha125/17-2 and NA226/3-1.

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