Title

Knob-Socket Predictions of Alpha-Helical Stability and Structure

Poster Number

17B

Lead Author Major

Biochemistry

Lead Author Status

Senior

Second Author Major

Biochemistry

Second Author Status

Senior

Third Author Major

Biochemistry

Third Author Status

Senior

Fourth Author Major

Biochemistry

Fourth Author Status

Senior

Format

Poster Presentation

Faculty Mentor Name

Jerry Tsai

Faculty Mentor Email

jtsai@pacific.edu

Faculty Mentor Department

Chemistry

Graduate Student Mentor Name

Taylor Rabara

Graduate Student Mentor Email

t_rabara@u.pacific.edu

Graduate Student Mentor Department

Chemistry

Additional Mentors

Graduate Mentor:

Melina Huey

m_huey@u.pacifc.edu

Chemistry

Abstract/Artist Statement

The Knob-Socket (KS) Model is a novel method to describe protein packing. In this work, the KS model is applied to predict intra-helical and inter-helical packing from 2° through 4° structures involving a four-residue tetrahedral motif. The model involves a four-residue tetrahedral motif called the Knob-Socket. The motif consists of a one amino acid knob from one 2° structure that can be packed into a three amino acid socket on another 2° structure. From an analysis of structures in the Protein Data Bank (PDB), the propensity of a set of three amino acids forming a socket was found to exist in 3 states: (1) free, without a knob and favoring intra-helical interactions, (2) filled, packed with a knob, favoring inter-helical interactions and (3) non, unpacked and disfavoring alpha-helical structure. From these propensities, a parallel, α-helical protein homodimer designated KSα1.1, was designed to validate the Knob-Socket Model. In previous research, single and double point mutations were introduced into the wild-type protein sequence. These mutations’ effect on helix content and stability correlated with the change in propensity of the six socket hexagon or Rabara surrounding a mutation. In this current research, additional single point mutations were introduced into the stable KSα-T14V/M20L mutant DNA. As an amino acid packing code, the calculated socket propensities allow for the prediction of the change in secondary structure content and stability to the KSα1.1-T14V/M20L protein. The mutant variants were created through site-directed mutagenesis and were analyzed through circular dichroism to further characterize the alpha-helicity of these mutants. The raw data collected from CD were then deconvoluted using DICHROWeb to quantify the alpha-helical content of each mutant. The alpha helical character of these mutants was then compared to the original predictions made from the change in propensities of the Rabara hexagon. An increase in alpha-helicity would indicate a more stable structure, whereas, a decrease indicates a less stable structure. To further characterize the stability of these structures, thermal and chemical denaturation studies were carried out for each mutant protein.

Location

DeRosa University Center Ballroom

Start Date

27-4-2018 10:00 AM

End Date

27-4-2018 12:00 PM

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Apr 27th, 10:00 AM Apr 27th, 12:00 PM

Knob-Socket Predictions of Alpha-Helical Stability and Structure

DeRosa University Center Ballroom

The Knob-Socket (KS) Model is a novel method to describe protein packing. In this work, the KS model is applied to predict intra-helical and inter-helical packing from 2° through 4° structures involving a four-residue tetrahedral motif. The model involves a four-residue tetrahedral motif called the Knob-Socket. The motif consists of a one amino acid knob from one 2° structure that can be packed into a three amino acid socket on another 2° structure. From an analysis of structures in the Protein Data Bank (PDB), the propensity of a set of three amino acids forming a socket was found to exist in 3 states: (1) free, without a knob and favoring intra-helical interactions, (2) filled, packed with a knob, favoring inter-helical interactions and (3) non, unpacked and disfavoring alpha-helical structure. From these propensities, a parallel, α-helical protein homodimer designated KSα1.1, was designed to validate the Knob-Socket Model. In previous research, single and double point mutations were introduced into the wild-type protein sequence. These mutations’ effect on helix content and stability correlated with the change in propensity of the six socket hexagon or Rabara surrounding a mutation. In this current research, additional single point mutations were introduced into the stable KSα-T14V/M20L mutant DNA. As an amino acid packing code, the calculated socket propensities allow for the prediction of the change in secondary structure content and stability to the KSα1.1-T14V/M20L protein. The mutant variants were created through site-directed mutagenesis and were analyzed through circular dichroism to further characterize the alpha-helicity of these mutants. The raw data collected from CD were then deconvoluted using DICHROWeb to quantify the alpha-helical content of each mutant. The alpha helical character of these mutants was then compared to the original predictions made from the change in propensities of the Rabara hexagon. An increase in alpha-helicity would indicate a more stable structure, whereas, a decrease indicates a less stable structure. To further characterize the stability of these structures, thermal and chemical denaturation studies were carried out for each mutant protein.