Structure/Function Analysis of S102P (and R550Q) Human

Cytochrome P450 Oxidoreductase (hPOR) Mutant(s)

Andrew Yang

Health Careers High School

March 5, 2014

Introduction: Protein StructureProtein primary

structure arises from the protein’s specific amino-acid sequence.

Amino acid sequence is dictated by the central dogma: DNA → mRNA→ Codon → Polypeptide (amino acid sequence)

Relating Structure with Function Depending on the chemical

properties of the amino acids in the primary structure, the protein folds into a specific secondary structure containing α helixes and β pleated sheets.

The protein’s actual 3-D conformation is dictated by the specific intermolecular interactions between amino acids in these α helixes and β pleated sheets.

Changes in tertiary structure can alter protein function by altering the amino acid interactions and thus changing the overall shape of the protein.

MutationsGenetic mutations occur when errors occur in DNA

replication or RNA transcription. These errors may change the amino acid sequence and alter protein conformation at the location of the mutation.

Protein function may be compromised, as illustrated in this figure below.

Human NADPH-Cytochrome P450 Reductase

• Human Cytochrome P450 Oxidoreductase (POR, shown below), is an enzyme in the liver and other organs that transfers electrons to the following cofactors, which are reduced as they accept electrons:

NADPH →→ FAD →→ FMN →→ Heme (Fe)•The heme group contains a central Iron atom which has high affinity for electrons.

Functions of POR Various other Cytochrome

P450s found throughout the body are dependent upon POR’s electron transfer to them.

These Cytochrome P450s are responsible for carrying out important processes such as drug metabolism and steroid synthesis, as shown in the figure to the right.

POR Mutations: Antley-Bixler Syndrome

Certain mutations of POR have been identified in human patients and classified based on the amino acid that has been altered.

These patients seem to exhibit symptoms of Antley-Bixler Syndrome, a rare genetic condition associated with a loss of function in POR in which craniofacial dysmorphism and/or altered steroidogenesis occurs as a result (Miller, 2004).

Human Skull

POR Mutations: Location in PORThe following figure shows the

locations of various mutations that have been identified in POR.

In this diagram, the FMN binding domain is violet, the FAD binding domain is blue, and the NADPH- binding domain is in green.

A green dot indicates that the mutation is “silent” and barely impacts POR function, while a red dot indicates that POR activity is less than 25% of the wild type POR activity. Note the approximate location of the highlighted S102P and R550Q mutations whose structure-function relationships are the subject of this study.

S102P R550Q

Impaired POR Function in Mutants Research in Dr. Masters’ Lab

indicates that binding affinity for FAD, a cofactor in electron transfer, is diminished in both the V492E and R457H POR human mutations. The human patients with these defects have impaired steroidogenesis and craniofacial defects.

In these two locations, the mutation causes changes in enzyme structure at the FAD binding site, resulting in enzyme instability and impairment of POR activity compared with the wild type protein as shown in this electron density diagram.

S102P and R550Q Mutations The S102P (serine to proline

substitution) and R550Q (Arginine to Glutamine) mutations were originally found in human subjects from the Czech Republic.

It is unknown whether S102P and/or R550Q impairs POR functionality, since these subjects are anonymous and, thus, any accompanying illness is not known.

For S102P, it is suspected that the proline substitution will disrupt the helical structure (creating a “kink”), disrupting the conformation of the protein in a crucial location in the molecule.

S102P R550Q

Objectives/HypothesisSince many mutations in proteins can be

innocuous, we propose to characterize the S102P and R550Q POR mutants for catalytic function as well as structural aberrations, compared with wild type POR. (Previously studied mutants were shown to have lowered FAD or FMN binding affinities by the Masters lab, suggesting that riboflavin therapy could be efficacious.)

It is expected that there will be a difference between the electron transfer rates of the S102P, R550Q mutants and the wild type POR when a cytochrome c reduction assay is performed. These studies are in progress.

Protein Expression In order to study the effects of

the mutation on the native functionality of POR, a fragment of human DNA, which codes for POR, is inserted into an E. coli plasmid vector to transfer the POR DNA into the bacteria to be synthesized into protein.

The E. coli cells are grown with shaking in broth media in 3-liter flasks.

The protein expressed by these cells is purified and collected for enzyme activity analysis (e.g., Cytochrome c reduction assay).

Transformation Experiments• Mutant POR is expressed by

the pET 28a vector E.coli plasmid, which contains an antibiotic resistance gene.

• The vector is inserted into E. coli BL-21 cells, and then the cells are plated with agar, which has the antibiotic corresponding with the antibiotic resistance gene in the vector.

• The resulting colonies appear as white dots on the agar plates and represent individual cells.

Growth and Induction

• The cells are bumped up to TB media at 37⁰C and grown in flasks for 24 hours on

a shaker.

• Growth is monitored by measuring absorbance of the media with a

spectrophotometer.

• When the cells are growing fastest at log phase with an absorbance of 0.6-0.8, a

chemical called IPTG is added; this process, called induction, allows the cells to

start expressing the protein of interest.

Experimental Plan

Harvesting

After induction, the cells were spun down in the centrifuge.

The resulting pellet was collected and frozen.After thawing, the pellet was homogenized with lysate

buffer to release the protein from the cells and centrifuged to rid the sample of cell debris, and the supernatant was collected for purification

Protein PurificationNickel Affinity Chromatography is a method of

obtaining purer protein. The proteins are “tagged” by adding additional histidine residues to the terminal end. The Ni column captures the histidines specifically and leaves untagged proteins behind.

Cytochrome c Reduction AssayThis diagram shows the

electron transfer mechanism occurring between wild type POR and its associated Cytochrome P450 from (NADPH → FAD → FMN → Heme group).

The Cytochrome c reduction assay measures electron transfer activity of POR through the absorbance changes in Cytochrome c, which is reduced and measured at 550 nm. Alternatively, activity of the associated Cytochrome P450 can be measured.

Acknowledgements

I would like to thank: My mentors: Dr. Satya Panda and Dr. Bettie Sue Masters, as

well as Ms. Karen McCammon and Ms. Katie Hinchee-Rodriguez for their guidance and assistance in this project.

My teacher: Ms. Catherine Gonzalez for teaching the

principles of scientific research.

ReferencesHart S, Wang S, Nakamoto K, Wesselman C, Li Y, Zhong X

(2008). Genetic Polymorphisms in cytochrome P450 oxidoreductase influence microsomal P450-catalyzed drug metabolism.

Hart S, Zhong X (2008). P450 oxidoreductase: genetic polymorphisms and implications for drug metabolism and toxicitiy.

Marohnic CC, Panda SP, Martasek P, Masters BS (2006). Diminished FAD Binding in the Y459H and V492E Antley-Bixler Syndrome Mutants of Human Cytochrome P450 Reductase. J Biol Chem 281: 35975-35982.

Xia C, Panda SP, Marohnic CC, Martasek P, Masters BS, Kim JP (2011). Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency. Proceedings of the National Academy of Sciences 108: 13486-13491.

Picture Referenceshttp://www.docstoc.com/docs/22467824/NADPH-Cytochrome-

P450-Oxidoreductasehttp://www.google.com/imgres?imgurl=&imgrefurl=http%3A

%2F%2Fwww.quora.com%2FHow-does-IPTG-induced-gene-expression-work-at-a-molecular-level

http://www.uky.edu/Pharmacy/ps/porter/CPR.htmhttp://www.chempep.com/ChemPep-Generic-Term_Protein.htmhttp://www.addgene.org/plasmid_protocols/

bacterial_transformation/http://www.bio-rad.com/en-us/product/protein-stainsAll other pictures and charts are either student-generated or

used with mentor permission