Rational Knob-Socket Predictions of Alpha-Helical Stability
Poster Number
13a
Introduction/Abstract
A construct that simplifies the complexity of residue packing would significantly impact our understanding and analysis higher order protein structure in the same way that the covalent peptide bond allows linear comparisons of protein sequences and main-chain hydrogen bonding patterns identify secondary structure. The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. Intra-helical interactions consist of a regular pattern of three residue triangular motifs called sockets, which contribute to helical stability. For inter-helical interactions, a single amino acid knob from one α-helix packs into a three amino acid socket within another α-helix. Therefore, sockets are defined in three categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. The three amino acid composition of a socket serves as a code that can be used to predict protein packing and by extension, can also be used to understand individual amino acid contributions to helical stability. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants have been rationally chosen to increase and decrease stability by relating KS propensities with changes to α-helical structure. In the KS α-helical model, each point mutation affects six surrounding sockets by altering the free/filled propensity values. By analyzing the changes in the propensities of these six sockets, KS based structure predictions were made for each mutant that relate to their stability. These predicted values are compared to the experimentally determined structure and stability of each protein from chemical and thermal denaturation studies as measured by circular dichroism spectroscopy. This study serves as a starting point to reveal how residue packing contributes to protein stability.
Purpose
Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. Intra-helical interactions consist of a regular pattern of three residue triangular motifs called sockets, which contribute to helical stability. For inter-helical interactions, a single amino acid knob from one α-helix packs into a three amino acid socket within another α-helix. Therefore, sockets are defined in three categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. The three amino acid composition of a socket serves as a code that can be used to predict protein packing and by extension, can also be used to understand individual amino acid contributions to helical stability.
Method
The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants have been rationally chosen to increase and decrease stability by relating KS propensities with changes to α-helical structure. In the KS α-helical model, each point mutation affects six surrounding sockets by altering the free/filled propensity values. By analyzing the changes in the propensities of these six sockets, KS based structure predictions were made for each mutant that relate to their stability. These predicted values are compared to the experimentally determined structure and stability of each protein from chemical and thermal denaturation studies as measured by circular dichroism (CD) spectroscopy.
Results
Over the length of KSα1.1 helix, over 10 point mutations were made. Predicted stabilities were calculated based on the change in KS propensities of the 6 effected sockets that are arranged in a hexagon around the point mutation. While most were predicted to decrease α-helicity, some were predicted to stabilize and increase helical character. Comparison of a deconvolution analysis of the CD spectra showed that the KS model was predictive. In addition, the double mutants were additive in their ability to stabilize the peptide into more α-helical content.
Significance
This study serves as a starting point to reveal how residue packing contributes to protein stability. The impact is that the work informs us on how to rationally predict the effect an amino acid mutation will have in a particular secondary structure environment. This data is useful for rational protein design as well as determining the effects of point mutations in disease states.
Location
DeRosa University Center
Format
Poster Presentation
Poster Session
Morning 10am-12pm
Rational Knob-Socket Predictions of Alpha-Helical Stability
DeRosa University Center
A construct that simplifies the complexity of residue packing would significantly impact our understanding and analysis higher order protein structure in the same way that the covalent peptide bond allows linear comparisons of protein sequences and main-chain hydrogen bonding patterns identify secondary structure. The novel knob-socket (KS) model provides a construct to interpret and analyze the direct contributions of amino acid residues to the stability in α-helical protein structures. Based on residue preferences derived from a set of protein structures, the KS construct characterizes intra- and inter-helical packing into regular patterns of simple motifs. Intra-helical interactions consist of a regular pattern of three residue triangular motifs called sockets, which contribute to helical stability. For inter-helical interactions, a single amino acid knob from one α-helix packs into a three amino acid socket within another α-helix. Therefore, sockets are defined in three categories: (1) free, unpacked and favoring intra-helical interactions, (2) filled, packed and favoring inter-helical interactions, and (3) non, unpacked and disfavoring α-helical structure. The three amino acid composition of a socket serves as a code that can be used to predict protein packing and by extension, can also be used to understand individual amino acid contributions to helical stability. The KS model was used in the de novo design of an α-helical homodimer, KSα1.1. Using site-directed mutagenesis, KSα1.1 point mutants have been rationally chosen to increase and decrease stability by relating KS propensities with changes to α-helical structure. In the KS α-helical model, each point mutation affects six surrounding sockets by altering the free/filled propensity values. By analyzing the changes in the propensities of these six sockets, KS based structure predictions were made for each mutant that relate to their stability. These predicted values are compared to the experimentally determined structure and stability of each protein from chemical and thermal denaturation studies as measured by circular dichroism spectroscopy. This study serves as a starting point to reveal how residue packing contributes to protein stability.
