Thermodynamic effects of proline introduction on protein stability.
The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.
Prajapati
Ravindra Singh
RS
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Das
Mili
M
Sreeramulu
Sridhar
S
Sirajuddin
Minhajuddin
M
Srinivasan
Sankaranarayanan
S
Krishnamurthy
Vaishnavi
V
Ranjani
Ranganathan
R
Ramakrishnan
C
C
Varadarajan
Raghavan
R
D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019277:Entropy; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C488923:LivJ protein, E coli; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; C028003:RbsB protein, E coli; D011993:Recombinant Fusion Proteins; C517609:TrxA protein, E coli; D013879:Thioredoxins; D011392:Proline