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

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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.


The enzymatic activity of wild type and some alanine insertion mutants. Enzymatic activity was monitored by absorbance change at 340 nm. (a) The reaction curves of TMP-sensitive mutants. (b) The reaction curves of some TMP-resistant mutants. (c) The reaction curves of the other TMP-resistant mutants. The reaction solution contains 5.56nM DHFR, 50 μM DHF and 60 μM NADPH, in 10mM potassium phosphate buffer C (pH 7.8). Reactions were performed at 20°C.
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f5-7_1: The enzymatic activity of wild type and some alanine insertion mutants. Enzymatic activity was monitored by absorbance change at 340 nm. (a) The reaction curves of TMP-sensitive mutants. (b) The reaction curves of some TMP-resistant mutants. (c) The reaction curves of the other TMP-resistant mutants. The reaction solution contains 5.56nM DHFR, 50 μM DHF and 60 μM NADPH, in 10mM potassium phosphate buffer C (pH 7.8). Reactions were performed at 20°C.

Mentions: We measured the enzymatic activity of several mutants to assess whether the TMP resistance assay is an adequate functional assay of DHFR. Figure 5 shows the reaction curves of enzymatic activity for wild type and the insertion mutants. It is clear that all the mutants expressed in the TMP-sensitive transformants have little or no activity, as shown in Figure 5(a), confirming that in these cases, the “Function Elements” determined by TMP resistance assay are valid. The reaction curves for the mutants expressed in the TMP-resistant transformants are shown in Figures 5(b) and (c). Some mutants are active (Fig. 5(b)), supporting the validity of the TMP resistance assay. However, the other mutants are essentially inactive (Fig. 5(c)). We hypothesize that these mutants maintain TMP binding ability but lose either catalytic activity or NADPH binding ability. Therefore, the TMP resistance assay can only evaluate the TMP binding ability, and is insufficient to identify the complete “Function Elements.”


Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase
The enzymatic activity of wild type and some alanine insertion mutants. Enzymatic activity was monitored by absorbance change at 340 nm. (a) The reaction curves of TMP-sensitive mutants. (b) The reaction curves of some TMP-resistant mutants. (c) The reaction curves of the other TMP-resistant mutants. The reaction solution contains 5.56nM DHFR, 50 μM DHF and 60 μM NADPH, in 10mM potassium phosphate buffer C (pH 7.8). Reactions were performed at 20°C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036773&req=5

f5-7_1: The enzymatic activity of wild type and some alanine insertion mutants. Enzymatic activity was monitored by absorbance change at 340 nm. (a) The reaction curves of TMP-sensitive mutants. (b) The reaction curves of some TMP-resistant mutants. (c) The reaction curves of the other TMP-resistant mutants. The reaction solution contains 5.56nM DHFR, 50 μM DHF and 60 μM NADPH, in 10mM potassium phosphate buffer C (pH 7.8). Reactions were performed at 20°C.
Mentions: We measured the enzymatic activity of several mutants to assess whether the TMP resistance assay is an adequate functional assay of DHFR. Figure 5 shows the reaction curves of enzymatic activity for wild type and the insertion mutants. It is clear that all the mutants expressed in the TMP-sensitive transformants have little or no activity, as shown in Figure 5(a), confirming that in these cases, the “Function Elements” determined by TMP resistance assay are valid. The reaction curves for the mutants expressed in the TMP-resistant transformants are shown in Figures 5(b) and (c). Some mutants are active (Fig. 5(b)), supporting the validity of the TMP resistance assay. However, the other mutants are essentially inactive (Fig. 5(c)). We hypothesize that these mutants maintain TMP binding ability but lose either catalytic activity or NADPH binding ability. Therefore, the TMP resistance assay can only evaluate the TMP binding ability, and is insufficient to identify the complete “Function Elements.”

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.