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Molten Globule Monomers in Human Superoxide Dismutase

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Molten Globule Monomers in Human Superoxide Dismutase Biuphysical Cherni.stry, 48 (1993) 171-182 171 Elsevier Science Publishers B.V., Amsterdam BIOCHE 01799 Molten globule monomers in human superoxide dismutase Norberto Silva, Jr. a,*, Enrico Gratton b, Giampiero Mei ‘, Nicola Rosato ‘, Ruth Rusch d and Alessandro Finazzi-Agrb ’ a Mayo CXnic, Guggenheim 14, 200 First St., SW Rochester, MN 55905 (USA) * Laborarory for Fluorescence Dynamics, Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, IL 61801 (USA) ’ Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Uniuersitri di Roma “Tar Vergata’: Ku 0. Raimondo 8, 00173 Rome (Italy) d Brown University, Biomedical Department, P.O. Box G, Prouidence, RI 02912 (USA) (Received 23 April 1993; accepted 13 July 1993) Abstract The time-resolved fluorescence decay and anisotropy of Cu/Zn human superoxide dismutase (HSOD) were studied as a function of temperature and denaturant concentration. In addition, circular dichroism (CD) measurements were performed on HSOD as a function of denaturant concentration in the amide and aromatic regions. The time-resolved fluorescence decay results reveal the existence of structural microhetero- geneity in HSOD. Furthermore, CD measurements and a global analysis decomposition of the time-resolved fluorescence decay over denaturant concentration shows the presence of an intermediate in the unfolding of HSOD by guanidinium hydrochloride. Considering our previous measurements of partially denatured HSOD as a function of protein concentration (Mei et al., Biochemistry 31 (1992) 72X-7230), our results strongly suggest that the unfolding intermediate is a monomer that displays a molten globule state. Keywords: Human superoxide dismutase; Time-resolved fluorescence; Global analysis; Conformational substates; Molten globule state 1. Introduction diates may be necessary for protein translocation across cell membranes [3]. Finally, knowledge of Protein folding is biologically important for protein folding could provide avenues toward de- many reasons. For instance, folding is necessary signing novel enzymes ‘. to initiate the function of various precursor en- Current research has established that some zymes [ll. Furthermore, protein folding interme- proteins exhibit an intermediate state during the folding/ unfolding process [4-71. Experimental methods confirming the existence of intermedi- ates include circular dichroism (CD), nuclear magnetic resonance, infrared spectroscopy and * Corresponding author. Fax (507) 284-9349. ’ Abbreviations: Circular dichroism (CD), human superoxide intrinsic viscosity measurements [8-111. In addi- dismutase (HSOD), guanidine hydrochloride (GdHCIJ, trypto- tion, another powerful method for investigating phan (Trp). protein folding intermediates is through the fluo- 0301-4622/93/$06.00 0 1993 - El sevier Science Publishers B.V. All rights reserved 172 Id Silva, Jr. et al. /Biophys. Chem. 48 0993) 17X-182 rescence decay of extrinsic and intrinsic probes molten globule state in HSOD. In addition, a (6, 11, 121. two-dimensional experiment has been performed Steady-state and time-resolved fluorescence to rigorously test the hypothesis of structural mi- studies have illuminated many differences be- croheterogeneity and the existence of a molten tween native and denatured proteins [12, 13-151. globule state. This involves time-resolved fluores- One general result from time-resolved fluores- cence decay experiments of HSOD over denatu- cence studies of denatured proteins is the in- rant concentration and temperature. The tem- creased heterogeneity in conformation as com- perature and denaturant range covered is rela- pared to the native state [15-171. This result has tively broad (1O’C to 50°C and 0.0 M to 6.5 M been confirmed by our studies on the single tryp- GdHCl) and the interval between experiments is tophan fluorescence decay of human superoxide relatively small (5°C and OS-l.0 M GdHCl). For dismutase (HSOD). HSOD is an ideal system to this work we plan to perform a global analysis of study protein denaturation because: (1) it con- the time-resolved fluorescence decay of HSOD tains a single Trp residue per monomer unit over denaturant concentration to test the exis- (HSOD is a dimer with identical subunits [18]), tence of an intermediate. Time-resolved fluores- which simplifies interpretive analysis, and (2) Trp cence anisotropy decay measurements were also is environmentally sensitive making it an ideal performed as a function of temperature and de- reporter of microheterogeneity. Our results have naturant concentration. The time-resolved fluo- shown that the time-resolved fluorescence decay rescence anisotropy measurements monitor the of denatured HSOD in 6.5 M guanidine hydro- “fluidity” of the environment of the Trp residue chloride (GdHCI) displays more heterogeneity in HSOD as a function of GdHCl concentration. than that of native HSOD [l]. These studies also showed that the native form of HSOD displays structural microheterogeneity. This is in agree- 2. Experimental ment with the concept of protein conformational 2.1. Sample preparation substates in systems such as myoglobin [19-211. Finally, our HSOD studies suggest that the Holo-HSOD was purified from human ery- monomerization stage of HSOD displays a molten throcytes [24]. Samples were dissolved in 0.01 M globule state. The molten globule state is a pro- potassium phosphate buffer at pH 7.6. Denatura- tein folding intermediate of considerable interest tion was accomplished by mixing an appropriate which is characterized as having native-like sec- amount of HSOD stock solution with ultra-pure ondary structure and fluctuating tertiary structure guanidine hydrochloride (from ICN Biomedical, [ll, 221.There is also theoretical evidence for the Inc., Cleveland, OH, USA). The final protein existence of a molten globule state in proteins concentration was around 30 FM giving an ap- WI. proximate optical density of 0.1 at 295 nm. The However, our previous evidence for a HSOD following concentrations of GdHCl were used: molten globule state were limited to experiments 0.0, 1.0, 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 at a single temperature (20°C). Furthermore, a M (time-resolved fluorescence anisotropy mea- full decomposition of the time-resolved fluores- surements were not done at 5.5 M GdHCl). At cence decay was not done with an intermediate in each denaturant concentration temperature de- mind. This decomposition is crucial if we are to pendent experiments were performed from 10°C suspect that the intermediate displays properties to 50°C in steps of 5°C. The temperature was which are characteristic of a molten globule state. monitored continuously during measurements by In addition, previous time-resolved fluorescence attaching a thermocouple to the sample cuvette. measurements were only correlated to the amide The readings of the thermocouple were moni- CD signal of HSOD. In this article we have tored by an Omega Digicator (from Omega Engi- extended the CD measurements to the aromatic neering, Stamford, CT, USA) with an accuracy of region which is a key test for the existence of a f O.l”C. N. Silva,Jr. et al. /Biophys. Chem. 48 (1993) 171-182 173 2.2. Time-resolved fluorescence and anisotropy fits are shown in three-dimensional graphs with measurements the x- and y-axes corresponding to temperature and denaturant concentration, respectively. The All time-resolved fluorescence and anisotropy temperature axis of these graphs correctly por- measurements were performed at the Laboratory trays the intervals between experiments (5°C). for Fluorescence Dynamics of the University of However, the GdHCl axis division does not com- Illinois at Urbana-Champaign, Urbana, IL, USA. pletely reflect the experiments conducted on Frequency domain techniques were used to mea- HSOD. In particular, no data was taken for 0.5, sure the fluorescence decay of all samples in the 1.5 and 2.5 M GdHCl for Figs. 2, 3, 4-6 and range of 10 to 240 MHz [25]. The light source was 11-13. In addition, no data was taken for 5.5 M a high repetition mode-locked Nd-YAG laser GdHCl in Figs. 4-6. In addition, some of the used to pump a rhodamine dye laser yielding a 10 data, being high in laser noise or having large ps pulse output of about 50 mW at 590 nm with a systematic error, were discarded. A total of 10 2 MHz repetition rate. This output was then experiments were removed from the time-re- frequency doubled to 295 nm and its harmonic solved fluorescence decay data set (Figs. 2,3 and content was used to excited the sample [26]. The 11-13) and a total of 12 experiments were re- excitation was polarized at the “magic angle” to moved from the time-resolved fluorescence an- eliminate rotational effects on the fluorescence isotropy decay data set (Figs. 4-6). As a result, an lifetime measurements [27]. The emission was interpolation was performed to fill in the missing observed using an optical filter combination of data points in all three dimensional graphs. The UV34 and U340 (from Oriel Corp., Stratford, corresponding interpolation points, (x,y>, for the CT, USA). rejected data sets are listed as follow for Figs. 2,3 and 11-13 where x is the temperature (in de- 2.3. Circular dichroism measurements grees Celsius) and y is the denaturant concentra- tion (in m/l): (15,4.5), (20,4.5), (20,5-O),(25,5.0), Circular dichroism measurements were carried (30,5.0), (45,5.0), (15,5.5), (20,5.5), (25,5.5) and out using a Jobin- Yvon CD VI spectropolarime- (25,6.0). For Figs. 4-6 the rejected data sets are: ter (from SPEX Industries, Inc., Edison, NJ, (45,3.5), (15,4.5), (20,4.5), (20,5.0), (25,5.0), USA). The sample holder was thermostated at (30,5.0),(45,5.0), (50,5.0) (25,6.0), (35,6.0), (40,6.0), 20°C using an external bath circulator. Measure- (50,6.0).
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