Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein.


Abstract

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein. Study holds ProTherm entries: 17404, 17405, 17406, 17407, 17408, 17409, 17410, 17411, 17412, 17413, 17414, 17415, 17416, 17417, 17418, 17419, 17420, 17421, 17422, 17423, 17424, 17425, 17426, 17427, 17428, 17429, 17430, 17431, 17432, 17433, 17434, 17435, 17436, 17437, 17438, 17439, 17440, 17441, 17442, 17443, 17444, 17445, 17446, 17447, 17448, 17449, 17450, 17451, 17452, 17453, 17454, 17455, 17456, 17457, 17458, 17459, 17460, 17461, 17462, 17463, 17464, 17465, 17466, 17467, 17468, 17469, 17470, 17471, 17472, 17473, 17474, 17475, 17476, 17477, 17478, 17479, 17480, 17481, 17482, 17483 Extra Details: mutant; stability; hydrogen bond

Submission Details

ID: iLcnrtLf3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Prajapati RS;Lingaraju GM;Bacchawat K;Surolia A;Varadarajan R,Proteins (2003) Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein. PMID:14635128
Additional Information

Number of data points 245
Proteins Maltose-binding periplasmic protein ; Maltose-binding periplasmic protein
Unique complexes 7
Assays/Quantities/Protocols Experimental Assay: dHcal pH:10.4 ; Experimental Assay: Tm pH:10.4 ; Experimental Assay: dHcal pH:9.9 ; Experimental Assay: Tm pH:9.9 ; Experimental Assay: dHcal pH:9.5 ; Experimental Assay: Tm pH:9.5 ; Experimental Assay: dHcal pH:9.0 ; Experimental Assay: Tm pH:9.0 ; Experimental Assay: dHcal pH:8.5 ; Experimental Assay: Tm pH:8.5 ; Experimental Assay: dHcal pH:8.0 ; Experimental Assay: Tm pH:8.0 ; Experimental Assay: dHcal pH:7.4 ; Experimental Assay: Tm pH:7.4 ; Experimental Assay: dHcal pH:7.0 ; Experimental Assay: Tm pH:7.0 ; Experimental Assay: dCp pH:6.5 ; Experimental Assay: dHcal pH:6.5 ; Experimental Assay: Tm pH:6.5 ; Experimental Assay: dCp pH:7.4, temp:63.0 C ; Experimental Assay: ddG ; Experimental Assay: Cm temp:37.0 C ; Experimental Assay: m temp:37.0 C ; Experimental Assay: dG_H2O temp:37.0 C ; Experimental Assay: Cm temp:30.0 C ; Experimental Assay: m temp:30.0 C ; Experimental Assay: dG_H2O temp:30.0 C ; Experimental Assay: Cm temp:22.0 C ; Experimental Assay: m temp:22.0 C ; Experimental Assay: dG_H2O temp:22.0 C ; Experimental Assay: Cm temp:14.0 C ; Experimental Assay: m temp:14.0 C ; Experimental Assay: dG_H2O temp:14.0 C ; Experimental Assay: Cm temp:10.0 C ; Experimental Assay: m temp:10.0 C ; Experimental Assay: dG_H2O temp:10.0 C ; Derived Quantity: dTm pH:10.4 ; Derived Quantity: dTm pH:9.9 ; Derived Quantity: dTm pH:9.5 ; Derived Quantity: dTm pH:9.0 ; Derived Quantity: dTm pH:8.5 ; Derived Quantity: dTm pH:8.0 ; Derived Quantity: dTm pH:7.4 ; Derived Quantity: dTm pH:7.0 ; Derived Quantity: dTm pH:6.5 ; Derived Quantity: ddG_H2O temp:37.0 C ; Derived Quantity: ddG_H2O temp:30.0 C ; Derived Quantity: ddG_H2O temp:22.0 C ; Derived Quantity: ddG_H2O temp:14.0 C ; Derived Quantity: ddG_H2O temp:10.0 C
Libraries Mutations for sequence KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK
Sequence Assay Result Units