Gavin King, Ph.D. - US grants

Intellectual Merit
In order for a cell to operate properly, some proteins must pass through membranes while other proteins must fold and insert themselves into membranes. How are these complex processes achieved at the molecular scale? What are the forces involved? How long does it take for the proteins to rearrange themselves? This research project aims to shed light on these issues by directly observing the arrangements and motions of individual proteins as they negotiate membranes. Techniques that are traditionally used in cellular studies have inherent challenges. As a result, there is little detailed knowledge regarding the complex molecular motions associated with these critical biological processes. The PI has developed and will employ a new tool- an ultra-high precision microscope- to directly study these interactions. This unique instrument, which is based on force, offers the potential to achieve atomically precise measurements of proteins and membranes in their natural state (in water at room temperature). These measurements, in conjunction with computer modeling and traditional biochemical experiments, will elucidate fundamental aspects of protein secretion and membrane protein folding.

Broader Impacts
The interface of molecular biology, nanotechnology and precision measurement provides a rich interdisciplinary medium that will broadly enhance the impact of this project. The educational and outreach activities of this project aim to address pressing needs across the educational spectrum, from elementary through graduate school to adult learning. These activities include: writing science education curriculum for a teacher training program aimed at improving elementary education in the state of Missouri; continued mentoring of high school and undergraduate students; creating incentives and training mechanisms to increase the impact of graduate student outreach activities; and entering direct dialogue with general audiences through interactive public speaking commitments. Collectively, these educational and outreach activities are designed to reach a broad range of individuals of all ages and backgrounds. Their overall objective is to promote scientific literacy and to convey the excitement of performing cutting edge scientific research.

Non-Technical:
The work under this award will reveal the fundamental principles of sequence and structure for pH-triggered and pH-insensitive peptides that cause macromolecule-sized destabilization of membranes, allowing passage of macromolecules at very low concentrations. This basic knowledge is currently lacking, and is a roadblock in the design of membrane-active peptides with broad applicability. The basic knowledge that will be gained through this work will be a significant advancement in the field of membrane-active peptides. The activity that will be studied here with this award, macromolecular-sized pore formation, has not been explored thus far and its fundamental principles and mechanism of actions are unknown. Yet, it has many potential applications in biotechnology and medicine. Success in this work may enable the rational design and preparation of membrane active peptides for drug delivery and biosensing, as well as for cancer therapies, anti-viral and anti-bacterial treatments, and in agriculture for the controlled and effective release of insecticides and fungicides at very low doses. The proposed research and outreach activities will promote interest in science, exchange of knowledge, and create synergistic interactions between students and researchers at different levels in different disciplines.

Technical:
The goal of this project is to characterize peptides that enable macromolecules to bypass the barrier of the plasma membrane. This collaborative effort by three investigators, an engineer, a physicist and a biochemist, will delineate the sequence-structure-function relationships of two families of peptides that form large macromolecule-sized pores in bilayers at low concentrations, one of which is triggered to act only at pH less than 6.0. While such behavior is extremely rare, or perhaps non-existent in nature, the investigators have discovered, by high-throughput screening, peptides with these properties. By comparing the sequences, functions and structures of these peptides, they will uncover the fundamental principles behind the unique activity of these families. The researchers will characterize the macromolecular permeabilization by pH-insensitive and pH sensitive pore formers by performing circular dichroism, fluorescence, and atomic force microscopy measurements. They will learn how the physical properties of the lipid bilayer affect the function of the two classes of peptides, and will test specific mechanistic hypotheses with sequence variations. The work will provide many research and outreach opportunities for graduate, undergraduate and high school students, launching many new careers in science. This award by the Biomaterials Program of the Division of Materials Research in the Directorate for Physical and Mathematical Sciences (the managing program), is co-funded by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.

Nontechnical:

An enormous challenge lies in developing nanoscale patterning methods to combine nanoelectronics with biological systems. Conventional patterning techniques have severe limitations when combining hard and soft materials. For example, electron beam lithography (EBL) can create patterns only a few nanometers in size, but requires sacrificial resists. These resists can be incompatible with biological materials and have environmental and safety concerns. Ice lithography is a novel technique that uses solid condensed gas instead of resist. This technique offers a path to atomically precise manufacturing to create nanophotonic and nanolectronic structures. There is, however, no such infrastructure in the United States. This conference will lay the foundations for a world-unique high precision facility that combines ice lithography with in-situ electron and scanning probe microscopy.

Technical:

The overarching goal of this multidisciplinary workshop is to bring together scientists and researchers from engineering, physics, chemistry, and biology in order to exchange ideas in recent innovations and trends in high resolution (below 5 nm) nanofabrication and characterization techniques. The workshop will highlight the science and technology at the cross-roads of quantum phenomena and bio-inspired materials. The natural self-assembly process, inherent to several classes of bio-inspired materials, has resulted in some of the most intriguing nanostructures. Tailoring the functionality of these structures remains a challenge that requires new processes for nanopatterning and nanofabrication. The workshop will provide a unique platform for researchers in academia and industry for envisioning a path forward towards advancing 3D nanofabrication, additive manufacturing, and nano-electronic devices using ice lithography. Additionally, the workshop will serve as a forum for integrating ideas on the synthesis and nanoscale characterization of functional nanomaterials and devices that seamlessly bridge condensed matter and biology. The workshop will lay the foundation for the establishment of future research infrastructure.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Proteins are important components of cells, which are the building blocks of all living things. Everything that enters and exits a cell (e.g., nutrients and pharmaceuticals) must go through membranes. Yet, little is known about how proteins and membranes interact with each other at the molecular scale. To better understand protein-lipid interactions, we will attach individual proteins to the sharp tip of an atomic force microscope, which is an extremely powerful microscope that is able to reveal interactions at the molecular scale. We will then bring these proteins into the proximity of membranes and use the microscope to record the resulting forces. Measuring these microscopic-level interactions will reveal details that depend on the specific proteins employed. More generally, the work will provide a deeper and quantitative understanding of fundamental activities that take place at the biological membrane. In terms of broader impacts, the project aims to improve STEM education and public science literacy. Several activities are planned to support these goals including developing and implementing a laboratory module for Missouri high school physics students; implementing and refining a new hands-on undergraduate course at the University of Missouri-Columbia targeted to non-science majors; and engaging in direct dialogue with general audiences in mid-Missouri through the Science Café, which is an informal venue that encourages questions from audience members.


The overarching goal of this research project is to elucidate interaction strengths, energy landscapes and kinetic pathways of model lipophilic proteins as they negotiate fluid lipid bilayers and thus to provide an improved and quantitative understanding of protein partitioning and folding at the lipid bilayer membrane interface. This will be achieved by combining high precision atomic force microscopy (AFM)-based force spectroscopy methodology with analytical modeling, computer simulations, and biochemistry. The central experimental observable (force) will be measured with state-of-the-art spatial-temporal precision. The project tackles fundamental questions in membrane biophysics such as: How do the protein sequence and the emergence of secondary structure affect the strength of a protein-lipid bilayer interaction? Three model protein systems of differing structural complexity will be employed in conjunction with supported lipid bilayers. Specific research goals include: (i) correlate peptide sequence with peptide-lipid bilayer interaction strength, energy landscape and kinetic pathway; (ii) deconvolve protein secondary structure from primary structure contributions in a model polypeptide-lipid bilayer interaction; and (iii) quantitatively characterize a critical peripheral membrane protein-lipid bilayer interaction underlying protein export activity in E. coli. By directly probing the repeated association and dissociation of model proteins from the lipid bilayer interface, this project is poised to provide an improved understanding of protein partitioning and folding in membranes, a fundamental biophysical process.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.