John Kuriyan, Professor

Closed (1) Mechanism of Raf Kinase Activation in Signal Transduction

Applications for fall 2021 are now closed for this project.

All cells must respond to external stimuli. In many cases signaling molecules incapable of diffusing through cellular membranes must be recognized at the plasma membrane but ultimately lead to changes in gene expression in nucleus.
This process known as signal transduction is carried out by cooperating protein receptors, switches, and enzymes. This project focuses on understanding the regulation of one such pathway called the mitogen-associated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signaling apparatus. Direct consequences of dysregulated MAPK/ERK signaling in multicellular organisms include oncogenesis, autoimmunity, and developmental defects. Combining studies in cells and in vitro, the primary goal of this work is to elucidate the mechanism of Raf kinase activation by Ras GTPases, one of the earliest events in MAPK/ERK signaling downstream of growth factor receptors.

Technically, the work for an undergraduate student could involve (1) the purification of recombinant protein from E. coli for in vitro reconstitution, (2) assembly of combinatorial mutagenesis libraries for high-throughput screens in mammalian cells, and/or (3) protein structure analysis from mutagenesis, molecular dynamics simulations, and cryogenic electron microscopy data.

Day-to-day supervisor for this project: Joseph Paul , Graduate Student

Qualifications: Enthusiasm for biology, chemistry, and protein structure and excellent communication skills are required. Prior experience with molecular biology, protein expression/purification, and/or programming (preferably Python or R) is desirable.

Weekly Hours: 12 or more hours

Related website: https://jkweb.berkeley.edu/

Closed (2) Bcr-Abl kinase domain mutations, drug resistance, and the road to understand kinase activity

Applications for fall 2021 are now closed for this project.

BCR-ABL is a constitutively active tyrosine kinase that causes chronic myeloid leukemia (CML). Imatinib mesylate is the first BCR-ABL tyrosine kinase inhibitor (TKI) developed to treat CML patients. However, mutations in the kinase domain (KD) of BCR-ABL led to imatinib-resistance in patients. As a result, second and third generation inhibitors are developed but patients keep acquiring new resistance mutations. The project involves deep mutagenesis of ABL KD, mainly the allosteric region, to investigate the effect of each mutation on drug resistance and allostery.

Mammalian cells (platE) will be transfected with the mutagenesis library to produce virus. Then IL-3 dependant murine Ba/F3 cells will be transduced with these viruses and the growth of the cells will be monitored in different conditions (-/+ IL-3, -/+ drugs etc). The genomic DNA will be isolated from the treated cells and high throughput sequencing will be used to study the effects of single/multiple substitutions.

Day-to-day supervisor for this project: Dr. Serena Muratcioglu , Post-Doc

Qualifications: As the project involves construction of a library and cell growth experiments any experience in basic cloning (PCR, transformation etc.) and mammalian cell culture (maintaining cells, transfection etc.) would be appreciated.

Weekly Hours: 12 or more hours

Related website: http://jkweb.berkeley.edu

Related website: https://jkweb.berkeley.edu/

Closed (4) Investigating DNA sliding clamp structural ensembles using simulation

Closed. This professor is continuing with Spring 2021 apprentices on this project; no new apprentices needed for Fall 2021.

Sliding DNA clamps and their associated clamp loaders are essential to all known living things, despite radical changes in other DNA replication machinery since the last universal common ancestor. Ring-like sliding DNA clamps tether polymerases to the DNA template, preventing frequent dissociation. This attachment turns the polymerase complex into a processive holoenzyme capable of replicating large DNA genomes. Clamp loaders are protein complexes which couple ATP binding and hydrolysis to large conformational changes in the clamp, first opening one interface of the ring, then closing it around DNA. We are separately developing a method to experimentally monitor the distance across a single sliding clamp interface as it is opened and closed by the loader. In this project, we will analyze enhanced sampling molecular dynamics simulations to understand the structural ensemble consistent with any distance measured by the experiment. We will also use this structural ensemble to estimate the thermodynamics of clamp opening and start additional simulations to characterize the paths connecting the open and closed states. While this project is entirely remote, a successful and interested apprentice could continue with related wetlab experiments in the future.


Day-to-day supervisor for this project: Kent Gorday, Graduate Student

Qualifications: The apprentice should be able to devote at least 9-12 hours per week to research and be excited about basic research. Candidates should be interested in learning basic programming and scientific computing skills. No experience is required.

Weekly Hours: 12 or more hours