Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for forcedependent accessibility of structure that makes use of a wireless Mini-Radio Centrifuge Force Microscope (MR.CFM) to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules were used to determine how the triple helix responds to force. As the primary load-and tension-bearing protein in vertebrates, the physical properties of collagen are of significant biomedical interest. How collagen's triple helix responds to applied force is controversial, with different studies inferring incompatible outcomes: overwinding, unwinding, shearing, or maintaining its zero-force structure. Because proteolytic cleavage requires a locally unwound triple helix, our experiments reveal how local collagen structure changes in response to applied force. Our first results show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix. The generality of this result will be discussed in the context of collagen's sequence heterogeneity.

acetyltransferase-related diseases. Therefore, I aim to engineer a lysine acetyltransferase (KAT) to have N-terminal acetyltransferase activity which will widen the activity of acetyltransferases. To accomplish this, I will compare and contrast the active sites of an N-terminal acetyltransferase (NAT) and a Lysine acetyltransferase (KAT) to generate a list of the differing amino acids, and then conduct mutagenesis on the KAT. Our goal is to ultimately convert a KAT into NAT, which will reveal information concerning which amino acids contribute to both the specificity and binding capabilities of the two acetyltransferases. Following this, we will compare and contrast our new functioning versions of the NAT to an archaeal N-terminal acetyltransferase with the goal of altering the archaeal NAT to be more like our new NAT versions generated before, which contain properties of the original KAT and NAT. The archeal NAT has a wide variety of substrates compared to other NATs. Using the same methods as above, we want to engineer a new version of the archaeal NAT that successfully acetylates both the N-terminal and lysine side chains, thereby producing the first acetyltransferase that is promiscuous enough to acetylate both substrates. By creating this ''promiscuous'' acetyltransferase, we will have created an acetyltransferase that could potentially become a molecular probe that could aid in the development of treatments of human diseases and certain types of cancer that are linked to acetyltransferases.

Cancer cells bypass cell death by changing the expression of the BCL-2 family of proteins, which are apoptotic pathway regulators. Upregulation of pro-survival BCL-2 proteins or downregulation of cell death effectors BAX and BAK interferes with the initiation of the intrinsic apoptotic pathway. In normal cells, apoptosis can occur through pro-apoptotic BH3-only proteins interacting and inhibiting pro-survival BCL-2 proteins. When cancer cells over-express pro-survival BCL-2 proteins, a potential remedy is the sequestration of these pro-survival proteins through a class of anti-cancer drugs called BH3 mimetics that bind in the hydrophobic groove of pro-survival BCL-2 proteins. To improve the design of these BH3 mimetics, the packing interface between BH3 domain ligands and pro-survival BCL-2 proteins was analyzed using the Knob-Socket model to identify the amino acid residues responsible for interaction affinity and specificity. A Knob-Socket analysis organizes all the residues in a binding interface into simple 4 residue units: 3-residue sockets defining surfaces on a protein that pack a 4th residue knob from the other protein. In this way, the position and composition of the knobs packing into sockets across the BH3/BCL-2 interface can be classified. A Knob-Socket analysis of 19 BCL-2 protein and BH3 helix co-crystals reveal multiple conserved binding patterns across protein paralogs. Conserved knob residues such as a Gly, Leu, Ala and Glu most likely define binding specificity in the BH3/BCL-2 interface, whereas other residues such as Asp, Asn, and Val are important for forming surface sockets that bind these knobs. These findings can be used to inform the design of BH3 mimetics that are specific to pro-survival BCL-2 proteins for cancer therapeutics.

The applications of protein design are limited by the ability to determine a protein's fold and function based solely on a given amino acid sequence. The novel knob-socket model accurately classifies protein tertiary packing structure and provides a code that aims to accurately describe the packing interactions of side-chain residues. It is able to classify protein structure based on distinct amino acid side-chain preferences, which then predict the knob-socket arrangement of the packed protein. From the frequency of appearance in known structures, the knob-socket model provides the basis for prediction of stabilizing and destabilizing mutations. This model was used to design a 27 amino acid sequence, KSalpha1.1, which has been shown to fold into a stable alpha-helix configuration and that oligomerizes. The effects of helix stability are characterized with CD spectroscopy and oligomerization with NMR. Using known correlations of amino acid composition to alpha-helix propensity from the knob-socket model specific amino acids in the KSalpha1.1 sequence are changed to predictively strengthened or weakened packing stability. Current work involves purification and characterization of these mutations in the KSalpha1.1 protein to demonstrate the accuracy of the knob-socket model for protein design. The results provide further proof of knob-socket's ability to correctly model the packing in protein structure and serve as a fundamental descriptor of higher order protein structure.

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