Membrane Proteins

Cytochrome Research

Membrane proteins present one of the principal challenges for structural biology today. Conventional methods of structure elucidation, i.e. X-ray crystallography and solution-state NMR, usually fail when applied to membrane proteins. Solid-state NMR spectroscopy is rapidly evolving as the method of choice for structure determinations of membrane proteins. The Ramamoorthy lab has played a central role in the development of solid-state NMR and is currently applying solid-state NMR to the structure elucidation of membrane proteins.

Cytochrome P450 - "Mother Nature's Blowtorch"

Catabolism of numerous endogenous and exogenous substances, including many drugs and toxins, is catalyzed by the cytochrome P450 family of enzymes. The scheme below shows the oxidation of cholesterol into a more soluble derivative; this is the first step in the degradation of cholesterol to bile acids.

Also shown in the scheme are the electron carrier proteins cytochrome b5 and Cytochrome P450 reductase which supply the two electrons needed by cytochrome P450 to perform its function.

The overall goal of this research is to better understand how Cytochrome P450 functions. New insight into this enzyme family's functionality can lead to better drug design to treat many diseases. In order to study this reaction, we are utilizing redox partners, such as Cytochrome P450 reductase, Cytochrome b5, and Cytochrome c, as well as membrane interactions.

Structural Biology of Full-length Cytochrome b5 and Cytochrome P450

Cytochrome b5 is an important electron carrier membrane associated protein whose intimate interactions with cytochrome P450 facilitate various catalytic oxidative reactions in the microsomes. This 16.7 kDa protein consists of three distinct regions: a water-soluble heme domain, a 14-residue linker and a transmembrane alpha-helix. The water-soluble domain of cytochrome b5 displays significant homology to both hemoglobin and myoglobin and can be easily cleaved off via trypsin. In its native form, the water-soluble domain of cytochrome b5 is anchored to the endoplasmic reticulum membrane via a hydrophobic transmembrane helix. This helix seems to play a critical role in the proper functioning of cytochrome b5 as evident in previous studies.

Some of the goals of this project include providing:

1) The high-resolution structures of Cytochrome b5 and Cytochrome P450 in magnetically aligned bicelles.

2) The high-resolution structure of Cytochrome b5:Cytochrome P450 complex in magnetically aligned bicelles.

3) Information on the dynamics of the Cytochrome b5 and Cytochrome P450, individually, in lipid bilayers.

4) Information on the dynamics and interactions of the Cytochrome b5:Cytochrome P450 complex in lipid bilayers.

5) Information on the dynamics and interactions of the Cytochrome P450 : Cytochrome P450 Reductase complex in lipid bilayers.

Structural Model of Full-length Cytochrome b5 and Cytochrome P450

A model of membrane-bound full-length cytochrome b5 and cytochrome P450 complex was obtained based on solution NMR data and site-directed mutagenesis studies. This model reveals complex formation between the acidic, convex surface of Cytochrome b5 and the basic, concave proximal surface of Cytochrome P450. The two complex structures in the model show the "hotspot" in the binding interface and also allow us to propose an inter-protein electron transfer pathway involving the highly conserved Arg125 on P450 serving as a salt bridge between the heme propionates of Cytochrome P450 and Cytochrome b5.

Complex structure (cluster I) Complex structure (cluster II)

Proposed electron transfer pathway (cluster I) Proposed electron transfer pathway (cluster II)

Structural Biology of Cytochrome c and and the FBD domain of Cytochrome P450 Reductase

Cytochrome P450 reductase is a diflavin protein that donates one to two electrons to Cytochrome P450 as well as a number of other cytochromes and proteins. There are four domains of this 78 kDa membrane associated enzyme which are: an N-terminal transmembrane domain, a flavin mononucleotide binding domain (FBD), a linker domain, and a FAD/NADPH domain. Electrons are donated to redox partners from the FBD binding domain.

Cytochrome c is a small, soluble protein that is integral in the electron transport chain, shuttling electrons between Cytochrome c reductase and Cytochrome c oxidase. Although Cytochrome c is a not a physiological redox partner of Cytochrome P450 reductase, it is a good model for studying the interaction and electron transfer between Cytochrome P450 and Cytochrome P450 reductase.

Some of the goals of this project include:

1. Solving the high-resolution structure of both cytochrome c and FBD in magnetically aligned bicelles.

2. Solving the high-resolution structure of the Cytochrome c:FBD complex in magnetically aligned bicelles.

Complex Structure (Cluster I) Complex Structure (Cluster II)

Thanks to NIH funding support!

Publications on this project

Publications on other membrane proteins the Ramamoorthy Lab has worked on in the past

Links for Cytochrome P450