KR100963305B1 - Method for analyzing denaturing processes between domains in protein - Google Patents

Abstract

The present invention relates to a method for analyzing the process of degeneration between domains in a protein, and more specifically, to denature the protein by using differential scanning calorimetry, circular dichroism (CD) and / or intrinsic fluorescence measurement. It is about analyzing the process.

Protein, domain, heat denaturation

Description

Method for analyzing denaturing processes between domains in protein

The present invention relates to a method for analyzing the process of degeneration between domains in a protein, and more specifically, to denature the protein by using differential scanning calorimetry, circular dichroism (CD) and / or intrinsic fluorescence measurement. It is about analyzing the process.

In general, differential scanning calorimetry (DSC) is a technique for analyzing the thermal denaturation process of a material by measuring the heat capacity according to temperature, and is used for thermostability analysis, melting point measurement, and thermodynamic parameter measurement. In the study of drugs, it is widely used for the thermal stability analysis of the preparation, and in the protein research, it is used for the thermal stability and domain structure analysis of proteins. However, if the protein's heat denaturation process is performed through a complex step without having a single denaturation process, data interpretation is difficult.

Differential scanning calorimetry is a widely applied method for thermostable and thermodynamic analysis of proteins, but it is difficult to interpret data when analyzing proteins consisting of two domains. The present technology provides a method for analyzing different domain denaturation processes in a protein consisting of two domains.

The present invention solves the above problems, and the object of the present invention is to provide a method for analyzing the degeneration process between domains in the protein.

In order to achieve the above object, the present invention

Provided is a method for analyzing protein denaturation characterized by using a combination of differential scanning calorimetry, circular dichroism (CD) and / or intrinsic fluorescence (Intrinsic Fluorescence).

In one embodiment of the present invention, the analysis method comprises analyzing the degeneration process between the domains of the protein, the protein domain is preferably two or more, in the present invention, for example, degeneration of a protein having two domains The process was analyzed.

In the present invention, the modification is preferably thermal modification, but is not limited thereto.

In addition, in one embodiment of the present invention, it is preferable to analyze the thermal denaturation process of the protein in the intact state.

In one embodiment of the invention, the analytical method can be applied, for example, to analyzing thermal denaturation of domains comprising alpha-helix structures, and to analyzing thermal denaturation of domains containing aromatic amino acids. Also applicable, but is not limited thereto.

The protein used in the assay method of the present invention was a cyclic AMP receptor protein (CRP).

Hereinafter, the present invention will be described.

Differential scanning calorimetry (DSC) is a technique that analyzes the thermal degeneration of a material by measuring its heat capacity over temperature. It is used for thermostability analysis, melting point measurement and thermodynamic parameters. In the study of drugs, it is widely used for the thermal stability analysis of the preparation, and in the protein research, it is used for the thermal stability and domain structure analysis of proteins. However, if the protein denaturation process does not have a single denaturation process but a complex step, data interpretation is difficult. In particular, if a protein has two different structural domains, the two domains may be thermally denatured at different temperatures, where differential scanning calorimetry data may show two or more different denaturing intervals or may be asymmetrical. Indicates. In this case, based on the structural information of the protein, the protein denaturation process can be specifically determined by using a combination of circular dichroism (CD) and intrinsic fluorescence (Intrinsic Fluorescence). In other words, when measuring circular dichroism according to temperature at 222 nm, thermal denaturation of domains richer in alpha helix structure can be selectively analyzed. The thermal denaturation of can optionally be analyzed. In the present invention, a sequential domain denaturation process was identified using a cAMP receptor protein (CRP, cyclic AMP receptor protein) as a model protein.

Cyclic AMP receptor protein (CRP) is a prokaryotic gene regulatory protein that is activated by cAMP binding. It is a dimer consisting of two identical subunits, and each subunit has two domains. The N-terminal domain binds cAMP and the C-terminal domain binds to DNA.

1 is differential scanning calorimetry measurement data of pure CRP protein not bound with cAMP. Seemingly as a single thermal denaturation process by showing a peak around about 65 ° C, the shape of the peak is somewhat asymmetrical and deconvolution into symmetrical peak elements. It can be seen that the sum of the two different symmetric peaks is fitting. Therefore, it means that two domains of CRP are denatured at different temperatures. Which domains are denatured first cannot be determined by differential scanning calorie data alone. To determine this, the thermodenatured region was measured by measuring circular dichroism and tryptophan intrinsic fluorescence.

Circular dichroism signals in the far-UV region reflect protein secondary structure information, especially at 222 nm, mainly due to alpha-helix. Therefore, the thermal denaturation data obtained from the circular dichroism signal according to the temperature at 222 nm can more selectively reflect the domain that is relatively rich in alpha helical structure among the two domains. In the case of CRP, the N-terminal domain is mainly composed of beta-sheet, but since the C-terminal domain is mainly composed of alpha helix, the circular dichroism data of FIG. It mainly reflects degeneration.

Intrinsic fluorescence of proteins is caused by aromatic amino acids such as tryptophan or tyrosine, and most of the fluorescence generated by excitation wavelength 280 nm is caused by tryptophan. Therefore, the data of the fluorescent signal of the protein excited at 280 nm according to the temperature can more selectively reflect the domain having the tryptophan of the two domains. In the case of CRP, since only the N-terminal domain has two tryptophans, the fluorescence data in FIG. 2 mainly reflects the thermal denaturation of the N-terminal domain.

As shown in FIG. 2, the CRP's thermal denaturation temperature is different when measured by the circular dichroism method and when measured by the intrinsic fluorescence analysis. This is the same as the two peak components shown by decomposition of the differential scanning calorimetry data at different temperature ranges. The thermal denaturation reflected by the circular dichroism is shown at a lower temperature than that reflected by the intrinsic fluorescence, which corresponds to the first low temperature peak component of the differential scanning calorimetry data, whereas the thermal denaturation reflected by the intrinsic fluorescence is second highest in the differential scanning calorimetry data. Match the temperature peaks factor. Therefore, it can be seen that the C-terminal domain is denatured first and then the N-terminal domain is denatured and the denatured sections of the two domains overlap at similar temperatures.

If the two domains were prepared separately and analyzed for heat denaturation, this could be different from the domain denaturation process in the intact protein. For example, if only the N-terminal domain of the CRP is prepared separately to measure the thermal denaturation, as shown in Figure 3 shows a completely different aspect from the intact CRP. In addition, even if the C-terminal domain of the CRP separately prepared by measuring the heat denaturation as shown in Figure 4 is completely different from the intact CRP. This can be interpreted as two domains interacting with each other in an intact CRP. Therefore, in order to analyze the different heat denaturation process of the two domains of the protein, it should not be measured as an independent domain but should be measured in an intact state. At this time, by applying the present technology, it is possible to find out the different thermal denaturation process of each domain in the intact protein state.

The thermal denaturation process of apo-CRP, considered a single cooperative transition in the present invention, was reinterpreted as an overlapping process of two individual transitions by each structural domain. The results show how spectroscopic methods such as circular dichroism and fluorescence can be effectively combined with a calorie approach to interpret the thermodynamic behavior of proteins from a structural point of view. By comparing the thermal behavior between the original domain and its isolated domain, the present invention indicates that in apo-CRP the two domains are strongly paired with each other by interactions including covalent as well as non-covalent interactions. When the domains are separated, the structure itself of each domain is very similar to that before the separation, so it can be seen that the interaction between domains strongly affecting the thermodynamic properties does not significantly affect the substructure of each domain. Therefore, the process of activating CRP by cAMP binding can be achieved through the existing network modification between domains.

In addition, the present invention can be applied to the analysis of the stability of the protein and the activity change analysis according to the denaturation, it can be applied to examining the stability and efficacy of protein drugs, and thus useful in the pharmaceutical industry for the development and production, preservation, etc. of protein drugs can do.

The present invention provides thermodynamic data confirming interactions between domains that occur in apo-CRP. To clearly interpret the complex thermal behavior of apo-CRP, spectroscopic methods for probing protein structures differently were adopted in conjunction with calorimetry. Then a qualitative or semi-quantitative comparison of the thermodynamic nature was undertaken between the original apo-CRP and its separate domains. We expect that the results will further facilitate biophysical studies to detail the interaction and structure of the apo-CRP domain in solution.

Hereinafter, the present invention will be further described through non-limiting examples.

Example  1: Protein Sample Preparation

Recombinant CRP was prepared by overproduction of E. coli strain BL21 (DE3) / pLysS comprising plasmid pT ₇ -CRP as described by Won, H.-S., et al. In Biochemistry 45, 13953-13962, 2000. . DNA fragments encoding 110-209 amino acid residues of the CRP to produce an isolated C-terminal domain were amplified by PCR using pT ₇ -CRP as a template and cloned into the pET-21a vector, resulting in E. coli BL21 Transformed into (DE3) / pLysS strain.

Cells expressing the proteins of interest were grown at 37 ° C. in M9 minimal medium and protein expression was induced by addition of IPTG (Sigma). Protein purification was performed by successive chromatography of Bio-Rex 70 (Bio-Rad), Blue Sepharose (Amersham), Hydroxyapatite (Bio-Rad), and Superdex 75 (Pharmacia). The isolated N-terminal domain is a limited protein of CRP purified with chymotrypsin with a slight modification of the methods of Blaszczyk and Wasylewski (Blaszczyk, U. and Wasylewski, Z. (2003) J. Prot. Chem. 22, 285-293) . Prepared by decomposition. 1.5 μM CRP solution in 20 mM potassium phosphate buffer (pH 7.0) containing 125 mM NaCl and 100 μM cAMP was treated with 5 μg / ml chymotrypsin (Boehringer-Mannheim) at 37 ° C. for 2 hours. The reaction was stopped by addition of 1 mM PMSF and the reaction mixture was subjected to continuous chromatography of Bio-Rex 70 and Superdex 75 for purification. Removal of residual cAMP bound to protein was performed by dialysis and confirmed by the value of A ₂₇₈ / A ₂₆₀ absorbance ratio. Protein concentration determination was determined using a known extinction coefficient (Cheng et. al ., (1993) Biochemistry 32, 8130-8139).

Example 2: Differential Scanning Calorimetry (DSC)

Calorimetry of 25 μM apo-CRP dissolved in standard buffer (50 mM potassium phosphate with 500 mM KCl, pH 6.7) additionally containing 0.2 mM DTT and 5% glycerol. , Heating rate was performed with a MicroCal VP-DSC microcalorimeter with 60 ° C / h. The data is origin provided by the DSC manufacturer.  The software was processed and analyzed using a baseline-subtracted and subtracted with a continuous baseline, as described by Orlov et al. (Orlov, VN, (1998) FEBS Lett . 433, 241-244) . The concentration normalized DSC curves were fitted into a non-two-state transition model.

Example  3: Circularly polarized light Dichroic ( CD Spectroscopy

Melted in standard buffer CD measurements of 4.0-6.0 μM protein were performed with a Jasco J-715 polar spectrometer with a temperature control using a quartz sample vessel of 0.2 cm diameter with 1 nm bandwidth and 4 s response time. To monitor thermal denaturation, CD signals at 222 nm were recorded every 0.2 ° C. with increasing temperature at 1 ° C./m from 20 ° C. to 80 ° C. to 95 ° C. The rate of variation ( F _d ) at a specific temperature (T) is defined as

Where CD _n and CD _d are the baseline values of the natural and denatured states, respectively, by extrapolating the data points ( CD _obs ) observed for the complete natural and fully denatured temperature ranges by linear least square fit. Got (Johnson et al ., (1995) Biochemistry 34, 5309-5316). To determine the denaturation midpoint, the apparent equilibrium constant K _app was calculated using the following equation.

The apparent midpoint, i.e., the intermediate denaturation temperature ( T _d ), is the temperature with half the rate of modification, i.e., F _d It was determined at the temperature (T) = 0.5 and thus ln K _app = 0. For this purpose, using the classic Worthof plot (Byrne, MP and Stites, WE (2007) Biophys. Chem . 125, 490-496), the absolute temperature of ln K _app for the range -2 <ln K _app <2 A temperature with ln K _app = 0 was determined by fitting a reciprocal of the linear function.

Example 4: Fluorescence Spectroscopy

Fluorescence emission of 1.5-3.0 μM protein dissolved in standard buffer was measured by increasing the temperature from 40 ° C. to 95 ° C. using a JASCO FP-6500 fluorescent spectrometer with a thermostat using a 10 mm diameter quartz sample vessel. The rate of temperature increase was taken at 60 ° C./h and the emission spectrum was recorded every 1 ° C. The excitation wavelength was 280 nm, the emission scan was 310 to 370 nm, and the scan speed was 2000 nm / min. To determine the intermediate denaturation temperature ( T _d ), the fluorescence intensity ratio ( I _{r, obs} ) is l _{max, d} (Maximum emission wavelength in the denatured state) and l _{max, n} The ratio of the fluorescence emission intensity at (maximum emission wavelength in the natural state) was calculated for each temperature. Then the variation ratio ( F _d ) is defined as

Where the baseline values I _{r , n} and I _{r , d} for the natural and denatured proteins are linear least square fits of the observed data points ( I _{r, obs} ) over the temperature range of the fully natural and fully denatured states, respectively. From extrapolation of. Finally, T _d was determined as described for the CD data.

The result of the above Example is as follows.

apo - CRP of DSC - Monitored  Heat denaturation

We first monitored the thermal denaturation of apo-CRP using DSC to demonstrate possible interactions between domains (FIG. 1). Thermal denaturation of CRP in the present invention showed an irreversible endothermic transition. Thermodynamic data of the present invention ( T _p = 65.1 ℃, DH = 130.9 kcal / mol) is Ghosaini et al. (Ghosaini, LR, Biochemistry 27, 5257-5261) ( T _m = 66.4 ± 0.1 ℃, DH _cal = 130.7 ± 6.0 kcal / mol). Although previous studies have addressed the thermal denaturation of apo-CRP as a single cooperative transition, the present invention has noted the asymmetric form of the transition and deconvolution into non-phase transition curves that can be applied to irreversible transitions. In FIG. 1 the denaturation curve of apo-CRP was well divided into overlapping individual transitions (or calorie domains): T _m = 64.0 ± 0.2 ° C. and DH _cal = 50.9 ± 8.8 kcal / mol and the second transition for the first transition. T _m = 65.3 ± 0.1 ° C and DH _cal = 80.0 ± 8.7 kcal / mol. DSC curves of proteins are often broken down into multiple components, representing separate transitions of individual domains (Zaiss, K. and Jaenicke, R. (1999) Biochemistry 38, 4633-4639). Thus, we predicted that the two components in FIG. 1 are due to the separate transition of each domain in apo-CRP. To confirm this possibility, the thermal denaturation of apo-CRP was further investigated by circular dichroism (CD) and fluorescence spectroscopy.

apo - CRP  Spectroscopy of denaturation monitoring

The data analyzed by CD of FIG. 2 can be effectively specified in the C-terminal domain composed mostly of alpha-helices rather than the N-terminal domain mainly occupied by the beta-screen structure during the denaturation process of the entire molecule. In contrast, the inherent fluorescence of the proteins reflects the microenvironment of the aromatic side chain, in particular the fluorescence emission with an excitation wavelength of 280 nm is characterized by tryptophan residues. Since CRP only has two tryptophan residues in the N-terminal domain, the fluorescence-monitored profile in FIG. 2 may specifically exhibit thermal denaturation of the N-terminal domain. If apo-CRP behaves as a single calorie domain, the two profiles will agree with each other (Byrne, MP and Stites, WE (2007) Biophys . Chem . 125, 490-496). However, the denaturation curve ( T _d = 62.9 ° C.) obtained from fluorescence significantly precedes the CD profile ( T _d = 66.1 ° C.) above 3 ° C. Based on the temperature range of denaturation and the T _d values, the fluorescence and CD curves correspond to the individual transition elements 1 and 2 degraded in the DSC profile of FIG. 1, respectively.

In summary, by mixing spectroscopic and calorimetric analyzes, the present invention confirmed that the thermal denaturation of apo-CRP can be separated and analyzed by individual domains, although strongly coupled by individual transitions. The transition of the N-terminal domain, which can be assigned to the fluorescence transition curve of FIG. 2 and the degraded DSC curve element 1 of FIG. 1, can be assigned to the CD transition curve of FIG. 2 and the DSC curve element 2 of FIG. 1. Occurs at lower temperatures than denaturation of the terminal domains. In order to investigate the possible interactions between the two domains in apo-CRP, the present invention analyzed the thermal behavior of each separately isolated domain compared to that of the original apo-CRP.

CRP Domain samples

Apo-CRP dimers are resistant to proteolysis, but are cleaved by a number of proteases in the presence of cAMP to produce dimers of N-terminal domains. These fragments, named aCRP, retain cAMP binding forces and their structure is identical to that of the corresponding parts in the original CRP. In particular, chymotrypsin treatment of CRP results in fragments (named CHαCRP) consisting of 1-136 residues in which the N-terminal domain is preserved. Thus, the present invention used CHαCRP as an isolated N-terminal domain of CRP. Since the C-terminal site is destroyed by enzymatic treatment, the isolated C-terminal domain was newly prepared as a recombinant protein. An isolated C-terminal domain (named βCRP ^C , 110-209 residues of the CRP) in a form comprising a C-helix originally has the same structure as that moiety in the CRP.

Separated CRP  Thermal denaturation of the domain

Although the structure of the domain itself does not change, separation of each domain can result in the loss of interaction between the domains that occur in the originally bound form, resulting in large changes in the thermodynamic and / or kinetic stability of the domain. In contrast, independent domains that do not interact with each other in their original form do not change their thermodynamic behavior even if covalent bonds between domains are destroyed. The results in FIGS. 3 and 4 clearly demonstrate that there is a strong cross-domain interaction in apo-CRP because the denatured profiles of apo-CHαCRP and βCRP ^C differ significantly from those of the original apo-CRP. Based on the CD profiles, apo-CH αCRP has a slightly wider denaturation temperature and shows about 5 ° C. increase in T _d than that of apo-CRP (FIG. 3). Fluorescence monitored denaturation of apo-CHαCRP ( T _d = 70.8 ° C.) is similar to the results by CD ( T _d = 71.3 ° C.), indicating that the protein behaves in a single calorie unit. This is the reason why minor differences occur due to non-phase transfer of proteins, system differences between CD and fluorescence probes (sensitivity, noise level, resolution, etc.). Thus, for the N-terminal domain transition (a DSC curve 1 in FIG. 1 and the fluorescence curve in FIG. 2) of apo-CRP, it can be concluded that the separation of the N-terminal domain increased its T _d above 7 ° C. The denaturation of βCRP ^C monitored by CD was very different from that of the original apo-CRP (FIG. 4). _{T d (T d = 71.3 ℃} ) similar to that of ^apo-C βCRP CHαCRP is compared with that of the original apo-CRP showed a significant increase. Comparing C-terminal domain transitions in apo-CRP (DSC curve 2 in FIG. 1 and CD curves in FIG. 2), separation of the C-terminal domain increased its T _d by at least 5 ° C. The temperature range of denaturation also showed a marked decrease in transition coordination, more than four times wider than that of apo-CRP or apo-CHαCRP.

In summary, the thermal behavior of isolated domains is characterized by a marked increase in T _d and reduced coordination, and is very different from that of the original apo-CRP before separation. As discussed above, the structure of the isolated domain coincides with the regions corresponding to the original apo-CRP. That is, the shape of each domain is not significantly disturbed by the breakdown of covalent bonds between the domains. Thus, when individual domains were separated, they exhibited significantly different thermal denaturation than the original form, not because of structural changes, but because the non-covalent interactions between the domains formed prior to separation were lost by separation. .

1 is a diagram showing the thermal denaturation of apo-CRP monitored by DSC. The solid line is the measured DSC signal, the thin line is the decomposed component 1, the thick line is the decomposed component 2, and the continuous circle is the sum of the two components. )

2 shows thermal denaturation of apo-CRP monitored by CD (continuous circle) and fluorescence (continuous black triangle) spectroscopy. The variation ratio ( F _d ) was calculated from the CD signal at 222 nm and the fluorescence emission ratio at l _{max , n} (342 nm) and l _{max , d} (334 nm), respectively.

3 shows thermal denaturation of apo-CHαCRP monitored by CD (continuous black dot) and fluorescence (continuous triangle) spectroscopy. For comparison, CD-monitored denatured profiles of apo-CRP are shown (continuous circles). The variation ratio ( F _d ) was calculated from the CD signal at 222 nm and the fluorescence emission ratio at l _{max , n} (342 nm) and l _{max , d} (334 nm), respectively.

4 shows thermal denaturation of βCRP ^C monitored by CD (continuous black dot) spectroscopy. For comparison, CD-monitored denatured profiles of apo-CRP are shown (continuous circles). The variation ratio ( F _d ) was calculated from the CD signal at 222 nm.

Claims (8)

In the method of analyzing the denaturation process of a protein having two or more domains, differential scanning calorimetry is measured for overall denaturation, the degeneration of the domain consisting of alpha helix is monitored by circular dichroism (CD), tryptophan A method for analyzing protein denaturation, characterized in that the denaturation of domains having residues is monitored by intrinsic fluorescence measurement and integratedly analyzed. delete delete The method of claim 1, wherein the denaturation is thermal denaturation. The method of claim 1, wherein the protein is intact. delete delete The method of claim 1, wherein the protein is a cyclic AMP receptor protein (CRP).

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