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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.


Examples of trimethoprim (TMP) resistance assay. The type of mutant contained in each transformant is shown in (a) and (d). Panels (b) and (e) show the colony formation of each transformant without TMP. Panels (c) and (f) indicate the colony formation of each transformant in media containing 1 μg/ml TMP.
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f1-7_1: Examples of trimethoprim (TMP) resistance assay. The type of mutant contained in each transformant is shown in (a) and (d). Panels (b) and (e) show the colony formation of each transformant without TMP. Panels (c) and (f) indicate the colony formation of each transformant in media containing 1 μg/ml TMP.

Mentions: TMP is a competitive inhibitor of DHFR. E. coli JM109 cells transformed with either wild type or alanine insertion mutants were streaked on agar plates containing 50 μg/ml ampicillin with and without TMP. TMP concentration was 1 μg/ml, if present. After 12-hour incubation, we measured colony formation (Fig. 1)12,21,22.


Systematic alanine insertion reveals the essential regions that encode structure formation and activity of dihydrofolate reductase
Examples of trimethoprim (TMP) resistance assay. The type of mutant contained in each transformant is shown in (a) and (d). Panels (b) and (e) show the colony formation of each transformant without TMP. Panels (c) and (f) indicate the colony formation of each transformant in media containing 1 μg/ml TMP.
© Copyright Policy
Related In: Results  -  Collection

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

f1-7_1: Examples of trimethoprim (TMP) resistance assay. The type of mutant contained in each transformant is shown in (a) and (d). Panels (b) and (e) show the colony formation of each transformant without TMP. Panels (c) and (f) indicate the colony formation of each transformant in media containing 1 μg/ml TMP.
Mentions: TMP is a competitive inhibitor of DHFR. E. coli JM109 cells transformed with either wild type or alanine insertion mutants were streaked on agar plates containing 50 μg/ml ampicillin with and without TMP. TMP concentration was 1 μg/ml, if present. After 12-hour incubation, we measured colony formation (Fig. 1)12,21,22.

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.