2009 SRI Participants-HHMI Undergraduate Program - Carnegie Mellon University

2009 Summer Research Institute (SRI) Participants

Turi Alcoser

Turi Alcoser, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Correlation of Enzymatic Activity and Structure in Glutathione Transferase

A study of the glutathione transferase (GST), responsible for metabolizing harmful chemicals in organisms, helps reveal a more detailed understanding of the relationship between protein dynamics and enzyme function. It is shown that the proline110 and leucine213 interaction in the GST structure plays an integral role in the specificity of the enzyme. Analysis when varying the substrates glutathione (GSH) and dinitrochlorobenzene (CDNB) with a spectrophotometer at a wavelength of 340 nm allows for calculation of specific activity of the wild type and mutants. Replacing the proline in the 110 residue with phenylalanine causes the specific activity of GST to be less than that of the wild type while mutation of the 110 residue with alanine makes the specific activity greater than that of the wild type.  Further analysis using nuclear magnetic resonance will allow for the structure dynamics of GST to be modeled.

Lianne Cohen

Lianne Cohen, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Nuclear Magnetic Resonance Spectroscopy of Fluorescent-dye-binding Single Chain Variable Fragments

Single chain variable fragments (scFV) are the smallest domain of an antibody that maintains its binding specificity, by connecting the variable light (VL) chain and variable heavy (VH) chain with a peptide linker. The isolated VL and VH can also bind to antigen, specifically fluorogens, dyes that fluoresce when bound to a scFV. By altering the proteins’ ability to bind to the dye, the scFVs act as biosensors. Their small size allows for rapid diffusion through tissue, providing for the detection of cancer cells. The structure of scFVs with and without dye enables further study of the dye binding affinity and conformational changes. In order to establish the proteins’ structures in solution, nuclear magnetic resonance (NMR) was employed. Codon optimization of one scFV was necessary for production in E. coli.

David Dobrowolski

David Dobrowolski, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Nuclear Magnetic Resonance Spectroscopy of Fluorescent-dye-binding Single Chain Variable Fragments

Single chain variable fragments (scFV) are the smallest domain of an antibody that maintains its binding specificity, by connecting the variable light (VL) chain and variable heavy (VH) chain with a peptide linker. The isolated VL and VH can also bind to antigen, specifically fluorogens, dyes that fluoresce when bound to a scFV. By altering the proteins’ ability to bind to the dye, the scFVs act as biosensors. Their small size allows for rapid diffusion through tissue, providing for the detection of cancer cells. The structure of scFVs with and without dye enables further study of the dye binding affinity and conformational changes. In order to establish the proteins’ structures in solution, nuclear magnetic resonance (NMR) was employed. Codon optimization of one scFV was necessary for production in E. coli.

Laura Filliger

Laura Filliger, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Investigation of the Interdependence Among Ribosome Assembly Factors, and their Effects on the Ribosomal Exit Tunnel Structure

A better understanding of the principles governing eukaryotic ribosome biogenesis provides a prototype to allow further study on how the pathway is carried out or disrupted in many diseases, including cancer. Assembly factors are an integral part of ribosome assembly as they serve to recruit necessary proteins. To identify the precise roles of assembly factors, we focused on the interdependence among this group of proteins and how they influence the structure of the ribosomal exit tunnel structure. Our experiments investigated two “B-cluster” proteins, Spb4 and Drs1, which are required for one specific step in maturation of the 60S subunit in yeast ribosomes: processing of the 27SB pre-RNA. Levels of the B-cluster proteins after the depletion of Spb4 were investigated. Drs1 was deleted in combination with Nop6 and Rrp14 to test for synthetic lethal interactions. In addition, we are interested in how assembly factors affect the mature ribosome structures. The Woolford lab has shown that the depletion of assembly factor Rlp7 prevents ribosomal proteins that form and surround the exit tunnel from associating with preribosomes. In order to expand the knowledge we have regarding the ribosomal proteins surrounding the exit tunnel, we aimed to analyze the ribosomal proteins Rpl24A and Rpl4B after the deletion of Rlp7. Our experiment outcomes will be described in our poster.

Siping He

Siping He, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Investigating the Role of Three GTPase Assembly Factors in the 60S Ribosome Subunit Assembly Pathway

Ribosomes are ancient highly conserved organelles whose proper construction is fundamental for cellular life because they carry out translation of mRNA into protein. It is no wonder then that the complex and largely unknown process of ribosome assembly has been a popular subject for research. The ribosome is composed of a large 60S subunit and a small 40S subunit in our model organism, Saccharomyces cerevisiae. Our research focuses on 3 GTPase assembly factors (Nog1, Drs1, and Nog2) which are involved in the construction of the 60S subunit. With Nog1, we investigated the effects of Nog1 depletion on ribosome assembly. TAP-tag purification process was used to extract the ribosomal complex post Nog1 depletion for analysis. Another goal was to N-terminally TAP-tag the Drs1 gene to allow for its isolation because attempts to C-terminally TAP-tag the gene have been unsuccessful. Finally, Nog2 is an essential GTPase assembly factor for which no mutant strain exists. Our goal was to construct a cold or temperature sensitive mutant nog2 strain of yeast to enable further investigation into its role in ribosomal assembly.

David Huang

David Huang, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Investigation of the Interdependence Among Ribosome Assembly Factors, and their Effects on the Ribosomal Exit Tunnel Structure

A better understanding of the principles governing eukaryotic ribosome biogenesis provides a prototype to allow further study on how the pathway is carried out or disrupted in many diseases, including cancer. Assembly factors are an integral part of ribosome assembly as they serve to recruit necessary proteins. To identify the precise roles of assembly factors, we focused on the interdependence among this group of proteins and how they influence the structure of the ribosomal exit tunnel structure. Our experiments investigated two “B-cluster” proteins, Spb4 and Drs1, which are required for one specific step in maturation of the 60S subunit in yeast ribosomes: processing of the 27SB pre-RNA. Levels of the B-cluster proteins after the depletion of Spb4 were investigated. Drs1 was deleted in combination with Nop6 and Rrp14 to test for synthetic lethal interactions. In addition, we are interested in how assembly factors affect the mature ribosome structures. The Woolford lab has shown that the depletion of assembly factor Rlp7 prevents ribosomal proteins that form and surround the exit tunnel from associating with preribosomes. In order to expand the knowledge we have regarding the ribosomal proteins surrounding the exit tunnel, we aimed to analyze the ribosomal proteins Rpl24A and Rpl4B after the deletion of Rlp7. Our experiment outcomes will be described in our poster.

Janet Lee

Janet Lee, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Investigation of the Interdependence Among Ribosome Assembly Factors, and their Effects on the Ribosomal Exit Tunnel Structure

A better understanding of the principles governing eukaryotic ribosome biogenesis provides a prototype to allow further study on how the pathway is carried out or disrupted in many diseases, including cancer. Assembly factors are an integral part of ribosome assembly as they serve to recruit necessary proteins. To identify the precise roles of assembly factors, we focused on the interdependence among this group of proteins and how they influence the structure of the ribosomal exit tunnel structure. Our experiments investigated two “B-cluster” proteins, Spb4 and Drs1, which are required for one specific step in maturation of the 60S subunit in yeast ribosomes: processing of the 27SB pre-RNA. Levels of the B-cluster proteins after the depletion of Spb4 were investigated. Drs1 was deleted in combination with Nop6 and Rrp14 to test for synthetic lethal interactions. In addition, we are interested in how assembly factors affect the mature ribosome structures. The Woolford lab has shown that the depletion of assembly factor Rlp7 prevents ribosomal proteins that form and surround the exit tunnel from associating with preribosomes. In order to expand the knowledge we have regarding the ribosomal proteins surrounding the exit tunnel, we aimed to analyze the ribosomal proteins Rpl24A and Rpl4B after the deletion of Rlp7. Our experiment outcomes will be described in our poster.

Celia Ludwinski

Celia Ludwinski, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Correlation of Enzymatic Activity and Structure in Glutathione Transferase

A study of the glutathione transferase (GST), responsible for metabolizing harmful chemicals in organisms, helps reveal a more detailed understanding of the relationship between protein dynamics and enzyme function. It is shown that the proline110 and leucine213 interaction in the GST structure plays an integral role in the specificity of the enzyme. Analysis when varying the substrates glutathione (GSH) and dinitrochlorobenzene (CDNB) with a spectrophotometer at a wavelength of 340 nm allows for calculation of specific activity of the wild type and mutants. Replacing the proline in the 110 residue with phenylalanine causes the specific activity of GST to be less than that of the wild type while mutation of the 110 residue with alanine makes the specific activity greater than that of the wild type.  Further analysis using nuclear magnetic resonance will allow for the structure dynamics of GST to be modeled.

Lillian Michalek

Lillian Michalek, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Correlation of Enzymatic Activity and Structure in Glutathione Transferase

A study of the glutathione transferase (GST), responsible for metabolizing harmful chemicals in organisms, helps reveal a more detailed understanding of the relationship between protein dynamics and enzyme function. It is shown that the proline110 and leucine213 interaction in the GST structure plays an integral role in the specificity of the enzyme. Analysis when varying the substrates glutathione (GSH) and dinitrochlorobenzene (CDNB) with a spectrophotometer at a wavelength of 340 nm allows for calculation of specific activity of the wild type and mutants. Replacing the proline in the 110 residue with phenylalanine causes the specific activity of GST to be less than that of the wild type while mutation of the 110 residue with alanine makes the specific activity greater than that of the wild type.  Further analysis using nuclear magnetic resonance will allow for the structure dynamics of GST to be modeled.


Gabe Ratliff, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Nuclear Magnetic Resonance Spectroscopy of Fluorescent-dye-binding Single Chain Variable Fragments

Single chain variable fragments (scFV) are the smallest domain of an antibody that maintains its binding specificity, by connecting the variable light (VL) chain and variable heavy (VH) chain with a peptide linker. The isolated VL and VH can also bind to antigen, specifically fluorogens, dyes that fluoresce when bound to a scFV. By altering the proteins’ ability to bind to the dye, the scFVs act as biosensors. Their small size allows for rapid diffusion through tissue, providing for the detection of cancer cells. The structure of scFVs with and without dye enables further study of the dye binding affinity and conformational changes. In order to establish the proteins’ structures in solution, nuclear magnetic resonance (NMR) was employed. Codon optimization of one scFV was necessary for production in E. coli.

Anna Romanova

Anna Romanova, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Investigating the Role of Three GTPase Assembly Factors in the 60S Ribosome Subunit Assembly Pathway

Ribosomes are ancient highly conserved organelles whose proper construction is fundamental for cellular life because they carry out translation of mRNA into protein. It is no wonder then that the complex and largely unknown process of ribosome assembly has been a popular subject for research. The ribosome is composed of a large 60S subunit and a small 40S subunit in our model organism, Saccharomyces cerevisiae. Our research focuses on 3 GTPase assembly factors (Nog1, Drs1, and Nog2) which are involved in the construction of the 60S subunit. With Nog1, we investigated the effects of Nog1 depletion on ribosome assembly. TAP-tag purification process was used to extract the ribosomal complex post Nog1 depletion for analysis. Another goal was to N-terminally TAP-tag the Drs1 gene to allow for its isolation because attempts to C-terminally TAP-tag the gene have been unsuccessful. Finally, Nog2 is an essential GTPase assembly factor for which no mutant strain exists. Our goal was to construct a cold or temperature sensitive mutant nog2 strain of yeast to enable further investigation into its role in ribosomal assembly.

Mariya Zakhalyavko

Maria Zakhalyavko, Carnegie Mellon University
Mentor: Dr. Gordon Rule

Investigating the Role of Three GTPase Assembly Factors in the 60S Ribosome Subunit Assembly Pathway

Ribosomes are ancient highly conserved organelles whose proper construction is fundamental for cellular life because they carry out translation of mRNA into protein. It is no wonder then that the complex and largely unknown process of ribosome assembly has been a popular subject for research. The ribosome is composed of a large 60S subunit and a small 40S subunit in our model organism, Saccharomyces cerevisiae. Our research focuses on 3 GTPase assembly factors (Nog1, Drs1, and Nog2) which are involved in the construction of the 60S subunit. With Nog1, we investigated the effects of Nog1 depletion on ribosome assembly. TAP-tag purification process was used to extract the ribosomal complex post Nog1 depletion for analysis. Another goal was to N-terminally TAP-tag the Drs1 gene to allow for its isolation because attempts to C-terminally TAP-tag the gene have been unsuccessful. Finally, Nog2 is an essential GTPase assembly factor for which no mutant strain exists. Our goal was to construct a cold or temperature sensitive mutant nog2 strain of yeast to enable further investigation into its role in ribosomal assembly.