Nanomedicine

Center for Protein Folding Machinery

Nanomedicine Challenges

Nanomedicine is an emerging field that seeks to engineer nanomachines and/or their interacting partners to generate specific biochemical functions for biotechnological applications, or to modify biological functions to correct errors in physiological processes inside living systems. These new nano-machines or interacting partners will be designed and fabricated using the same physical and chemical principles as those of naturally occurring molecules and must be physiologically compatible with other naturally occurring molecular machines in a living system. The challenges of making such a new nanomachine and interacting partners operational and effective require the application of multiple methodologies in medicine, biology, chemistry, physics, computing and engineering sciences in an integrated and coordinated manner.

We will not be able to accomplish such a goal with one or two specialists as is normally achieved in a R01 type of research project. We have to understand the action of the targeted nanomachine and interacting partners at the molecular, cellular and organismal levels in such a way that we can measure, manipulate and predict its biochemical or biological behaviors. We have to use physical methods to make measurements to discover the design principles in a conformationally dynamic nanomachine; computational methods to design in silico a nanomachine or its partners with new functionality; biochemical and engineering methods to fabricate it experimentally first in vitro and then in an authentic biological environment; and to cycle iteratively among measurements, engineering designs, experimental implementations and biological efficacy assays before a final nanomachine product with new functionality can emerge.

Throughout the project, we expect improvements of existing technologies as well as inventions of new experimental or computational methodologies by our team of investigators, who are driven by the overarching goals of the project. A human challenge in this project is to have our investigators, who are used to engaging in an individual research style, to adopt a highly coordinated team approach, to share data openly and to integrate measurements or computational outputs among different investigators in a pipeline of sophisticated and correlated investigative processes. Another challenge will be to work out a fair formalism to credit individual investigators so that their career development and peer recognition would not be compromised in the traditional norms of our academic institutions.

Goals and Approaches

How proteins fold in the cell is a fundamental question in modern biology, but a poorly understood one. The transformation of one-dimensional genetic information into three-dimensional protein structures depends on the accuracy and efficiency of the process of protein folding, often called "the second half of the genetic code". While small proteins with simple chain topologies can fold spontaneously, the vast majority of cellular proteins are unable to reach the native state without the assistance of elaborate cellular nanomachines composed of proteins known as molecular chaperones.

Eukaryotic cells contain a chaperonin complex named TRiC (~1MDa). TRiC is essential for viability based on its role in folding a number of eukaryotic proteins, such as actin, tubulin and the VHL tumor suppressor that cannot be folded by other cellular chaperones. We will focus our effort on the mammalian chaperonin TRiC because of its high biomedical relevance. In parallel, we will also use the archaeal chaperonin Mm-cpn from M. maripaludis, which has been found to have a high structural and sequence similarity to TRiC and is thus a good model system to understand and modify chaperonin function. Unlike TRiC, which is a hetero-oligomer, Mm-cpn is homo-oligomeric and offers the advantage of straightforward genetic modification via bacterial expression.

Our long term goal is to use chaperonins as nanomachines to direct the folding and unfolding of proteins of biomedical and biotechnological interest. Our aims will include both in vitro and in vivo applications. Our first thrust is to engineer chaperonins to target specific proteins of our choice. For instance, chaperonin variants will be optimized to fold proteins of pharmacological importance, to promote folding of mutant tumor suppressors, and to refold crystallins in the eye lens. The last example has the potential to become a therapeutic treatment for cataracts. Our second thrust is to harness the activity of native cellular chaperonins so as to inhibit folding of improperly activated proteins (e.g., oncogenes) and sequester folding intermediates, such as those of aggregation-prone proteins that underlie neurodegeneration (e.g., huntingtin and A-beta peptide). This goal will be achieved by using designed adaptor molecules that target the protein of interest to the chaperonin. Furthermore, by engineering regulated chaperonin-targeting domains that can be masked/unmasked using small molecules, it may be possible to apply chaperonin-mediated folding/unfolding as a switch to turn biological functions on and off. Finally, since chaperonins are nano-devices with large central cavities that can open and close their lids in a regulated manner, we plan to ultimately develop and engineer them as versatile nano-containers for a variety of ligands, including proteins, RNA, and semiconductor particles. These trigger-controlled containers will have many applications, including delivery of drugs and other ligands to cells as well as the fabrication of biological nanomaterials.

To achieve our goals in engineering a chaperonin and/or substrate for therapeutic and biotechnological applications, it is necessary to have detailed knowledge of (i) the key interactions between the chaperonin and substrate during the folding event as well as (ii) the dynamic properties of the subunits and domains of the chaperonin during the folding cycle. Our team integrates biochemical, genetic, spectroscopic, biophysical, computational, and engineering techniques for maximum synergy and impact.

Our biophysical approach will employ novel imaging methods that can capture structures of the chaperonin in action, not only in vitro but also in vivo inside the cell. The imaging tools will include electron cryomicroscopy (cryoEM), electron cryo-tomography, single-molecule fluorescence microscopy and trapping, X-ray solution scattering and crystallography. Though these are relatively advanced technologies, we will need to improve them to cope with the complexity of the TRiC with the substrates because of their dynamic properties. In addition, we will explore biophysical measurements combined with computation to visualize folding within the cell using single-molecule imaging and electron cryo-tomography. We plan to combine and integrate biochemical, spatial and temporal measurements of chaperonin and other related nanomachines inside a single cell to map the cellular factors affecting the chaperonin-assisted folding process. This research approach is challenging and unproven; we believe it will succeed given the diverse expertise of the team.

In silico folding of a small protein in an isolated solvent environment is an active research area. However, the substrate proteins of interest in our study are generally large, on the order of tens of kDa. Our computational approach will be to develop new tools that utilizing the structural and chemical data generated from experimental measurements to predict the folding pathway of the substrate, such as the VHL suppressor protein. We will also employ computation to explore the environmental factors that influence the dynamics of the chaperonin during the folding process. Success on these fronts would clearly represent a significant advance in our ability to predict the native structures of proteins. Our predictions will be tested experimentally by a variety of biophysical methods as well as more traditional biochemical techniques. Due to the novelty of these attempts and the computational challenges involved, we will gain a great deal of valuable information that would suggest improvements for the next generation of computational methods.

Nanomedicine: Unique and Distinct

Our nanomedicine approach reflects fundamental differences in the function of the TRiC machinery, compared with enzymes catalyzing small molecule substrates. The TRiC protein-folding functions cannot be understood or controlled with models based on two or three conformational states. Rather this eight subunit cyclic complex almost certainly requires multiple subunit motions utilizing a large set of distinct conformations, reflecting stages of the chaperone reaction pathway for folding critical intracellular proteins. Its substrates, which fail to fold spontaneously, must be guided through multiple obligatory partially folded intermediate conformations within the chaperone nanocycle. The cryoEM, spectroscopic and computational tools being developed here are required to identify the transient but absolutely critical subunit motions and conformational states of the machinery and its substrates. Knowledge of some of these currently unknown states opens up the possibility of selecting or designing agents to inhibit, promote, or change the specificity of the chaperone machinery. This nanomedicine approach opens an important pathway to the development of new therapeutic agents for human protein misfolding and protein deposition disease.

The eventual goal of our NDC is to modulate protein folding pathways and chaperone function for therapeutic and biomedical applications. We have assembled a team of 15 investigators from 6 institutions with expertise in chaperones, protein folding, cryoEM, crystallography, single-molecule imaging and trapping, computer simulation and modeling, engineering and translational medicine. The team gathers together resources and expertise that could not be found in a single-investigator group. By directing the efforts of the team onto a single problem we will be able to obtain results outside of the scope of regular research projects. In addition, we will seek additional collaborators in medicine and biotechnology that will benefit from our research approaches and missions to explore unanticipated research opportunities. The organization, collaborations and communications in our NDC exemplify the 21st century goal of conducting interdisciplinary research via new mechanisms of data sharing, analysis and design in multiple institutional sites and disciplines.

Our Center can be a unique intellectual resource to other NDC who need assistance in solving protein folding and aggregation problems. In general, nanomachines must undergo conformational variations during their physiological cycles. Our imaging, trapping, and computational tools will be suited not only to study chaperonins but also any other nanomachine system of comparable complexity. Thus, our proposed research will help overcome significant technical barriers for a number of nanomedicine applications in other NCD.

As part of the activities in our NDC proposal, we plan to develop a didactic course in “Analysis and Design Principles of Biological Nanomachinery”. This course will be conducted with cyber tools by our investigators in different institutions. This course is aimed at bridging the gap between biology and engineering for students initially in our 6 participating institutions and eventually accessible to all other NDC institutions. Our long term goal is to expose a large pool of talented engineering students to nanomedicine concepts so that they then go on to contribute to this emerging cross-disciplinary field.

Investigators

PI: Wah Chiu (Baylor College of Medicine)

Co-PIs: Judith Frydman (Stanford U), Paul Adams (LBNL), Steve Chu (LBNL), Scott Delp (Stanford U), David Gossard (MIT), Eric Jonasch (UT MD Anderson Cancer Center), Jonathan King (MIT), Tanja Kortemme (UCSF), Michael Levitt (Stanford U), Steven Ludtke (Baylor College of medicine), W.E. Moerner (Stanford U), Vijay Pande (Stanford U), Andrej Sali (UCSF), Huda Zoghbi (Baylor College of Medicine).

Up to Top