Limits...
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.


Example of the solubility assay with fluorescent staining of SDS-PAGE. a, wild type, b, 1A2, c, 112A113 and d, 113A114. Lane 1, whole-cell; 2, precipitant; 3, supernatant. The left lane of each panel is the molecular weight marker, and the corresponding molecular weights are given.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036773&req=5

f2-7_1: Example of the solubility assay with fluorescent staining of SDS-PAGE. a, wild type, b, 1A2, c, 112A113 and d, 113A114. Lane 1, whole-cell; 2, precipitant; 3, supernatant. The left lane of each panel is the molecular weight marker, and the corresponding molecular weights are given.

Mentions: Figure 2 shows typical examples of the results of SDS-PAGE followed by fluorescent stain. The deeply stained band is due to DHFR. Whole-cell lysate was also subjected to PAGE to confirm expression of the mutant. For wild type and 1A2, the fluorescent intensity of the DHFR band of supernatant is much higher than that of the precipitant, indicating that these are soluble. On the other hand, the precipitant DHFR band only shows fluorescence for 112A113 and 113A114, indicating that these are insoluble. The ratio of the fluorescence intensity of the precipitant to that of the total fluorescent intensity was examined for all possible alanine insertion mutants. Figure 3 shows a characteristic pattern. There are several regions on the primary structure that exhibit a high precipitant ratio, similar to the result of TMP resistance assay. We assume that a region with a high precipitant ratio is essential for structure formation, and that alanine cannot be inserted in these regions.


Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase
Example of the solubility assay with fluorescent staining of SDS-PAGE. a, wild type, b, 1A2, c, 112A113 and d, 113A114. Lane 1, whole-cell; 2, precipitant; 3, supernatant. The left lane of each panel is the molecular weight marker, and the corresponding molecular weights are given.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036773&req=5

f2-7_1: Example of the solubility assay with fluorescent staining of SDS-PAGE. a, wild type, b, 1A2, c, 112A113 and d, 113A114. Lane 1, whole-cell; 2, precipitant; 3, supernatant. The left lane of each panel is the molecular weight marker, and the corresponding molecular weights are given.
Mentions: Figure 2 shows typical examples of the results of SDS-PAGE followed by fluorescent stain. The deeply stained band is due to DHFR. Whole-cell lysate was also subjected to PAGE to confirm expression of the mutant. For wild type and 1A2, the fluorescent intensity of the DHFR band of supernatant is much higher than that of the precipitant, indicating that these are soluble. On the other hand, the precipitant DHFR band only shows fluorescence for 112A113 and 113A114, indicating that these are insoluble. The ratio of the fluorescence intensity of the precipitant to that of the total fluorescent intensity was examined for all possible alanine insertion mutants. Figure 3 shows a characteristic pattern. There are several regions on the primary structure that exhibit a high precipitant ratio, similar to the result of TMP resistance assay. We assume that a region with a high precipitant ratio is essential for structure formation, and that alanine cannot be inserted in these regions.

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.