Browsing by Subject "Specificity"

Now showing 1 - 5 of 5

Conformational dynamics plays a significant role in HIV reverse transcriptase resistance and substrate selection
(2012-12) Nguyen, Virginia Myanh; Johnson, Kenneth A. (Kenneth Allen)
Human immunodeficiency virus reverse transcriptase (HIV RT) is a virally encoded polymerase responsible for replicating the HIV genome. Most HIV treatments include nucleotide RT inhibitors (NRTIs) which inhibit HIV RT replication by serving as a substrate for the polymerase reaction but then blocks subsequent polymerization after incorporation. However, resistance to these NRTIs may occur through specific mutations in HIV RT that increase the discrimination of HIV RT for natural nucleotides over NRTIs. The role of enzyme conformational dynamics in specificity and substrate selection was studied using transient kinetic methods on HIV RT enzymes that have been site-specifically labeled with a conformationally sensitive fluorophore, to measure the rates of binding and catalysis. First, HIV RT with the mutation of lysine to arginine at the residue position 65 (K65R) was examined for its resistance against the NRTI tenofovir diphosphate (TFV), an acyclic deoxyadenosine triphosphate (dATP) analog. It was found that HIV RT K65R resistance to TFV was achieved through decreased rates of catalysis and increased rates of dissociation for TFV over dATP when compared with the kinetics of wild-type HIV RT. Moreover, global fitting analysis confirmed a mechanism where a large conformational change, after initial ground state binding of the substrate, contributed significantly to enzyme specificity. This led to our investigation of the molecular basis for enzyme specificity using HIV RT as a model system. Again, transient kinetic methods were applied with the addition of molecular dynamics simulations. The simulated results were substantiated by the corroborating experimental results. It was found that a substrate-induced conformational change in the transition of HIV RT from an open nucleotide-bound state to a closed nucleotide-bound state was the major determinant in enzyme specificity. The molecular basis for substrate selection resulted from the molecular alignments of the substrate in the active-site, which induced the conformational change. When the correct nucleotide was bound, optimal molecular interactions in the active-site yielded a stably closed complex, which promoted nucleotide incorporation. In contrast, when an incorrect nucleotide was bound, the molecular interactions at the active-site were not ideal, which yielded an unstable closed complex, which promoted substrate dissociation rather than incorporation.
Engineering and investigation of protease fine specificity
(2010-12) Li, Haixin; Georgiou, George; Iverson, Brent; Hoffman, David; Robertus, Jon; Ren, Pengyu
The Escherichia coli (E. coli) outer membrane protease OmpT is an endopeptidase of the omptin family in gram negative bacteria. OmpT cleave preferentially between two consecutive basic residues, especially Arg-Arg, and it has been classified as an aspartyl protease based on its crystal structure although biochemical confirmation of a catalytic aspartyl residue is lacking (Vandeputte-Rutten, et al., 2001). Our lab has successfully engineered the P1 and P1’ specificity and selectivity of OmpT by employing novel strategies for the isolation of enzyme variants that cleave desired substrates from large combinatorial libraries screened by flow cytometry. However, the engineering of proteases with altered specificity beyond the P1 and P1’ residues of the substrate have not been demonstrated. By applying high throughput screening of large libraries of OmpT constructed by structure-guided saturation mutagenesis of the S2 subsite (which recognizes the P2 residue), as well as random mutagenesis by error prone viii PCR and DNA shuffling, we engineered an OmpT variant exhibiting about 56 fold change in the selectivity for the P2 position in peptide substrates. Specifically, this enzyme preferred an acidic residue (Glu) over Tyr which is preferred by the wild type OmpT. Molecular modeling was then employed to provide insights on how mutations in OmpT mediated this change in P2 specificity. A long term goal of protease engineering is to generate highly specific enzyme variants that can be used for the irreversible inactivation of disease targets. The anaphylatoxin C3a is a key mediator in inflammation and has been implicated with multiple inflammatory diseases. Since the site of anaphylatoxin C3a recognized by cellular receptors lie in its C-terminus, a protease cleaving the C-terminus of C3a could be therapeutically relevant. Using high throughput screening and directed evolution we successful isolated C3a cleaving enzyme variants and have characterized them biochemically. Finally as part of this dissertation we have employed high throughput screening methods to dissect the substrate specificity of members of the kallikrein family of mammalian proteases which are implicated in a number of physiological and disease functions. The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction and vascular cell growth. KLK6 is over-expressed in breast and ovarian cancer tissues and has been shown to cleave peptides derived from human ix myelin protein and the Aβamyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6 by substrate phage-display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin-and chymotrypsin-like selectivities with Tyr/Arg preferred at the P1 site, Ser/Arg strongly preferred at P1’ and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1’. Docking simulations of consensus peptide substrates was used to infer possible identities of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates such as ionotropic glutamate receptor (GluR) and synphilin.
Kinetic characterization of CRISPR-Cas12a DNA targeting
(2021-05-06) Strohkendl, Isabel Suzanna; Russell, Rick, 1969-; Finkelstein, Ilya J; Johnson, Arlen W; Johnson, Kenneth A; Taylor, David W
Cas12a is a type V class 2 CRISPR-Cas nuclease that has recently been repurposed as a precision DNA-cutting tool, alongside the type II Cas9. Their potential for biotechnological and genome editing applications has driven enthusiastic efforts to biochemically characterize their assembly and RNA-guided DNA targeting mechanisms. Two important obstacles that must be resolved before these nucleases can be used widespread are their specificity and efficiency; CRISPR nucleases are known to cut sequences that resemble their intended target, and within the cell—where our genomic DNA is packaged into protein-rich chromatin—it is unclear how target preferences are influenced. Here, we quantitatively study Acidaminococcus sp. Cas12a DNA targeting behavior in vitro using a kinetics-based approach. We begin by characterizing Cas12a binding and cleavage on matched and mismatch targets (Chapter 2), uncovering how Cas12a achieves such high levels of specificity despite cleaving DNA in a rate-limiting binding regime. We then characterize Cas12a DNA targeting on substrates that begin resemble genomic chromatin (Chapter 3). Lastly, we review the fundamentals of CRISPR nuclease specificity and how engineering efforts to develop a ‘high-fidelity’ nuclease should be focused (Chapter 4).
Sequencing effects on the acquisition and retention of general knowledge and detailed memory specificity
(2018-10-04) Noh, Sharon Mina; Preston, Alison R.; Lewis-Peacock, Jarrod A
Using a category learning paradigm, we measured the acquisition and retention of generalized (category-level) and detailed (exemplar-level) knowledge as a function of different learning experiences to improve different types of knowledge (general vs. specific). Participants were trained to identify paintings by different artists for which half of the artists were studied in a blocked sequence and the remainder were interleaved. Participants were tested on general (category) and more detailed (exemplar) knowledge, both immediately after training and after a 1-week delay. We found that although memory performance declines over time, generalized knowledge is more stable. Our results also suggest that generalization and specificity may be competing processes: on immediate test, the schedule that is better for general knowledge (interleaved) is worse for detailed memory, and vice versa. Collectively, our results can inform theories of learning and memory and help determine the optimal conditions for enhancing long-term retention in various contexts.
Specific response generation and elaborative discourse structure
(2022-05-09) Ko, Wei-Jen; Li, Junyi Jessy; Durrett, Greg; Choi, Eunsol; Ji, Yangfeng
When composing text, a writer has to carefully choose the discourse structure for coherence and effective communication. For each sentence, this involves deciding how much detail to provide (specificity), and what details to elaborate on. The goal of the thesis is to move towards better discourse structure in natural language generation by understanding how to control specificity in generation and what details to provide. The first part of the thesis explores techniques to control the specificity in dialogue response generation and tackles the plausibility issues that arise as more specific sentences are generated. The second part explores what to be specific about. We create question datasets which we apply to modeling the elaborative discourse structure between sentences, and question answering of openended questions.