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Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase

View Article: PubMed Central - PubMed

ABSTRACT

Decoding sequence information is equivalent to elucidating the design principles of proteins. For this purpose, we conducted systematic alanine insertion analysis to reveal the regions in the primary structure where the sequence continuity cannot be disrupted. We applied this method to dihydrofolate reductase (DHFR), and examined the effects of alanine insertion on structure and the enzymatic activity by solubility assay and trimethoprim resistance, respectively. We revealed that DHFR is composed of “Structure Elements”, “Function Elements” and linkers connecting these elements. The “Elements” are defined as regions where the alanine insertion caused DHFR to become unstructured or inactive. Some “Structure Elements” overlap with “Function Elements”, indicating that loss of structure leads to loss of function. However, other “Structure Elements” are not “Function Elements”, in that alanine insertion mutants of these regions exhibit substrate- or inhibitor-induced folding. There are also some “Function Elements” which are not “Structure Elements”; alanine insertion into these elements deforms the catalytic site topology without the loss of tertiary structure. We hypothesize that these elements are involved essential interactions for structure formation and functional expression. The “Elements” are closely related to the module structure of DHFR. An “Element” belongs to a single module, and a single module is composed of some number of “Elements.” We propose that properties of a module are determined by the “Elements” it contains. Systematic alanine insertion analysis is an effective and unique method for deriving the regions of a sequence that are essential for structure formation and functional expression.

No MeSH data available.


Solution structures of some alanine insertion mutants and the comparison with the solubility assay. (a) Far-UV CD spectra of wild type and alanine insertion mutants. Curves 1–6 represent wild type, 73A74, 85A86, 25A26, 47A48 and 74A75, respectively. DHFR concentration is 0.2mg/ml in 10mM potassium phosphate buffer C (pH 7.8) at 20°C. (b) Ellipticity at 203 nm is plotted against the precipitant ratio shown in Figure 4. The ellipticity is almost 0 for the folded DHFR, whereas it is large and negative for unstructured DHFR.
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f4-7_1: Solution structures of some alanine insertion mutants and the comparison with the solubility assay. (a) Far-UV CD spectra of wild type and alanine insertion mutants. Curves 1–6 represent wild type, 73A74, 85A86, 25A26, 47A48 and 74A75, respectively. DHFR concentration is 0.2mg/ml in 10mM potassium phosphate buffer C (pH 7.8) at 20°C. (b) Ellipticity at 203 nm is plotted against the precipitant ratio shown in Figure 4. The ellipticity is almost 0 for the folded DHFR, whereas it is large and negative for unstructured DHFR.

Mentions: In order to confirm that a mutant with high precipitant ratio is unstructured, we randomly selected mutants to examine their solution structures by CD. Figure 4(a) shows examples of CD spectra of wild type and some alanine insertion mutants. Wild type, 73A74 and 85A86 have low precipitant ratios (lower than 40%). 47A48 and 74A75 have precipitant ratios higher than 60%, and 25A26 has a medium ratio (50%). We can conclude that 73A74 and 85A86 take native conformation, although the spectral shapes are slightly different from that of wild type. On the other hand, the CD spectra of 47A48 and 74A75 are typical spectra for denatured structures. The difference may be due to the loss of the exciton coupling of two tryptophans observed in the wild type26. The CD spectrum of 25A26 possesses both structured and unstructured properties. The CD values at 203 nm are plotted against the precipitant ratio in Figure 4(b). From the figure, we set the boundary between being foldable and unfoldable at a precipitant ratio of 60%. The alanine-insertion sites that break protein tertiary structure form contiguous regions in the primary sequence. We assumed that these regions undergo interactions that are essential in order to form the tertiary structure. We can derive 12 such regions: region 1, I2-V10; region 2, L28-E48; region 3, I60-L62; region 4, W74-I82; region 5, E90-G96; region 6, G97-P105; region 7, K106-H114; region 8, E120-G121; region 9, D127-Y128; region 10, E129-P130; region 11, W133-S135; and region 12, Y151-E157. These regions are shaded in Figure 3.


Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase
Solution structures of some alanine insertion mutants and the comparison with the solubility assay. (a) Far-UV CD spectra of wild type and alanine insertion mutants. Curves 1–6 represent wild type, 73A74, 85A86, 25A26, 47A48 and 74A75, respectively. DHFR concentration is 0.2mg/ml in 10mM potassium phosphate buffer C (pH 7.8) at 20°C. (b) Ellipticity at 203 nm is plotted against the precipitant ratio shown in Figure 4. The ellipticity is almost 0 for the folded DHFR, whereas it is large and negative for unstructured DHFR.
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Related In: Results  -  Collection

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f4-7_1: Solution structures of some alanine insertion mutants and the comparison with the solubility assay. (a) Far-UV CD spectra of wild type and alanine insertion mutants. Curves 1–6 represent wild type, 73A74, 85A86, 25A26, 47A48 and 74A75, respectively. DHFR concentration is 0.2mg/ml in 10mM potassium phosphate buffer C (pH 7.8) at 20°C. (b) Ellipticity at 203 nm is plotted against the precipitant ratio shown in Figure 4. The ellipticity is almost 0 for the folded DHFR, whereas it is large and negative for unstructured DHFR.
Mentions: In order to confirm that a mutant with high precipitant ratio is unstructured, we randomly selected mutants to examine their solution structures by CD. Figure 4(a) shows examples of CD spectra of wild type and some alanine insertion mutants. Wild type, 73A74 and 85A86 have low precipitant ratios (lower than 40%). 47A48 and 74A75 have precipitant ratios higher than 60%, and 25A26 has a medium ratio (50%). We can conclude that 73A74 and 85A86 take native conformation, although the spectral shapes are slightly different from that of wild type. On the other hand, the CD spectra of 47A48 and 74A75 are typical spectra for denatured structures. The difference may be due to the loss of the exciton coupling of two tryptophans observed in the wild type26. The CD spectrum of 25A26 possesses both structured and unstructured properties. The CD values at 203 nm are plotted against the precipitant ratio in Figure 4(b). From the figure, we set the boundary between being foldable and unfoldable at a precipitant ratio of 60%. The alanine-insertion sites that break protein tertiary structure form contiguous regions in the primary sequence. We assumed that these regions undergo interactions that are essential in order to form the tertiary structure. We can derive 12 such regions: region 1, I2-V10; region 2, L28-E48; region 3, I60-L62; region 4, W74-I82; region 5, E90-G96; region 6, G97-P105; region 7, K106-H114; region 8, E120-G121; region 9, D127-Y128; region 10, E129-P130; region 11, W133-S135; and region 12, Y151-E157. These regions are shaded in Figure 3.

View Article: PubMed Central - PubMed

ABSTRACT

Decoding sequence information is equivalent to elucidating the design principles of proteins. For this purpose, we conducted systematic alanine insertion analysis to reveal the regions in the primary structure where the sequence continuity cannot be disrupted. We applied this method to dihydrofolate reductase (DHFR), and examined the effects of alanine insertion on structure and the enzymatic activity by solubility assay and trimethoprim resistance, respectively. We revealed that DHFR is composed of “Structure Elements”, “Function Elements” and linkers connecting these elements. The “Elements” are defined as regions where the alanine insertion caused DHFR to become unstructured or inactive. Some “Structure Elements” overlap with “Function Elements”, indicating that loss of structure leads to loss of function. However, other “Structure Elements” are not “Function Elements”, in that alanine insertion mutants of these regions exhibit substrate- or inhibitor-induced folding. There are also some “Function Elements” which are not “Structure Elements”; alanine insertion into these elements deforms the catalytic site topology without the loss of tertiary structure. We hypothesize that these elements are involved essential interactions for structure formation and functional expression. The “Elements” are closely related to the module structure of DHFR. An “Element” belongs to a single module, and a single module is composed of some number of “Elements.” We propose that properties of a module are determined by the “Elements” it contains. Systematic alanine insertion analysis is an effective and unique method for deriving the regions of a sequence that are essential for structure formation and functional expression.

No MeSH data available.