Stereochemical Criteria for Prediction of the Effects of Proline Mutations on Protein Stability

When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The φ dihedral angle of Pro is constrained to values close to −65° and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle φ and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design

Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design

Structural Correlates of the Temperature Sensitive Phenotype Derived from Saturation Mutagenesis Studies of CcdB

Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4

A <i>dPIP5K</i> Dependent Pool of Phosphatidylinositol 4,5 Bisphosphate (PIP₂) Is Required for G-Protein Coupled Signal Transduction in <i>Drosophila</i> Photoreceptors

<div><p>Multiple PIP₂ dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP₂ supporting these events has been proposed, the mechanism by which they are generated is unclear. In <i>Drosophila</i> photoreceptors, the hydrolysis of PIP₂ by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP₂ re-synthesis in photoreceptors. Loss of <i>dPIP5K</i> causes profound defects in the electrical response to light and light-induced PIP₂ dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP₂ synthesis following light induced PIP₂ depletion. Other PIP₂ dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking <i>dPIP5K</i> function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP₂ required for normal <i>Drosophila</i> phototransduction. Our results define the existence of multiple pools of PIP₂ in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.</p></div

<i>dPIP5K¹⁸</i> photoreceptors have normal ultrastructure and unaltered levels of transduction proteins.

<p>(A) TEM images showing the ultrastructure of control (i-ii), <i>dPIP5K³⁰</i> (iii-iv) and <i>dPIP5K¹⁸</i> (v-vi). The cross sectional view of a single ommatidium (i, iii, v) and a high magnification view of a single rhabdomere (ii, iv, vi) are shown for each genotype. (B) Optical neutralization images of <i>dPIP5K¹⁸</i> retinae showing normal rhabdomere ultrastructure in flies grown in a 12h L/D cycle as well as in 24 hrs constant light. Images shown are from flies aged nine days post-eclosion. (C) Western blot analysis of head extracts from wild type, <i>dPIP5K¹⁸</i>, <i>dPIP5K³⁰</i> probed with antibodies to each of the major phototransduction proteins. The antibodies used are indicated at the right side of each panel. Tubulin is used as loading control for each set of blots. (D) Single optical transverse sections of a control and <i>dPIP5K¹⁸</i> retina probed with antibodies to Rhodopsin (Rh1) (i, ii) and TRP (iii, iv).</p

Subcellular localization of different PIPKs in adult photoreceptors.

<p>(A) Western blot showing localization of dPIP5K in different sub-cellular fractions prepared from adult Drosophila heads. Fractions shown are: HL-total head lysate; C-cytoplasm; N, nuclear; M-microsomal/membrane. An antibody to INAD is used as a membrane marker. Histone 1 has been used as a nuclear marker and Tubulin as a marker for cytosol. (B) Confocal image showing the distribution of endogenous dPIP5K as detected by a polyclonal antibody from wild type and <i>dPIP5K¹⁸</i> photoreceptors. The enrichment of dPIP5K staining at the rhabdomere membrane (arrow) in wild type is missing in <i>dPIP5K¹⁸</i>. (C) Double staining experiments on wild type retinae showing the co-localization of dPIP5K (green) with Rh1 (red). (D) Localization of dPIP5K overexpressed from its endogenous genomic locus. Confocal image from retinae of control flies and those overexpressing dPIP5K using Rh1-GAL4. The protein is shown localized to the rhabdomere membrane. (E) Confocal image showing localization of overexpressed dPIP4K in adult <i>Drosophila</i> photoreceptors. Phalloidin staining marking the rhabdomeres shown in green and dPIP4K localization detected by antibody labeling shown in red. Merged image shows that dPIP4K is excluded from the rhabdomeres.</p