2007 — 2010 |
Fenton, Aron W |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Interpreting Conformational Differences in An Allosteric Protein, Pyruvate Kinas
Allosteric Regulation; Binding; Binding (Molecular Function); Biological; CRISP; Computer Retrieval of Information on Scientific Projects Database; Data; Environment; Funding; Global Change; Grant; Institution; Investigators; L-phenylalanyl-L-alanine; Mammals, Rabbits; Molecular; Molecular Interaction; Multienzyme Complexes; Muscle; Muscle Tissue; NIH; National Institutes of Health; National Institutes of Health (U.S.); Oryctolagus cuniculus; Phe-Ala; Property; Property, LOINC Axis 2; Proteins; Pyruvate; Pyruvate Kinase; Pyruvates; Rabbit, Domestic; Rabbits; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Source; System; System, LOINC Axis 4; United States National Institutes of Health; analog; design; designing; enzyme complex; experiment; experimental research; experimental study; gene product; improved; inhibitor; inhibitor/antagonist; phenylalanylalanine; phosphoenol transphosphorylase; phosphoenolpyruvate kinase; research study; response; social role
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0.958 |
2008 — 2019 |
Fenton, Aron W |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dissecting Allostery in Pyruvate Kinase @ University of Kansas Medical Center
? DESCRIPTION (provided by applicant): Project Summary In both type 1 and type 2 diabetes, hepatic secretion of glucose is a primary contributor to hyperglycemia. In turn, hyperglycemia is the root of many chronic complications associated with these diseases. Our recent in vivo data using transgenic mice confirm the potential of activating human liver pyruvate kinase (hL-PYK) to counteract the hyperglycemia associated with diabetes. To facilitate future allosteric drug designs to activate (or prevent inhibition of) hL-PYK in the treatment of hyperglycemia, the long-range goal of our laboratory is to characterize the molecular/atomic level mechanism of allosteric activation of hL-PYK by (Fru-1,6-BP), allosteric inhibition by alanine, and inhibition b protein phosphorylation. However, drugs that resemble native effectors or other biological compounds are likely to also bind other proteins and cause side-effects. As an alternative, we will now characterize areas of the hL-PYK protein outside of native effector binding sites that have roles in allosteric mechanisms and, thus, can be targeted in future rational design of allosteric drugs that do not resemble biological compounds. A primary innovation in our approach to studying allosteric mechanisms is recognition of the potential of divorce allosteric regulation from ligand binding. Therefore, we will distinguish which ligand-induced changes in the protein contribute to ligand binding and which play roles in allosteric regulations. In Aim 1 w will monitor and probe to identify which protein-substrate interactions have roles in allosteric functions, using difference attenuated total reflectance infrared spectroscopy (ATR-IR) to monitor bond changes in the substrate (we will NOT evaluate protein with IR), a substrate analogue series, a divalent metal cation series, site-directed random mutagenesis of substrate contact residues, mutant cycle analysis, and loop modifications. Effector binding will be monitored using 2nd derivative absorption spectroscopy. In turn, the binding of effector will be evaluated over a concentration range of substrate or a substrate analogue as a means of determining allosteric coupling. In Aim 2, we will use hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) and X-ray crystallography to evaluate conformational and dynamic changes among the various enzyme complexes that define the allosteric energy cycles for each regulation (Fru-1,6-BP, alanine, and phosphorylation). In Aim 3, we will initiate a study to generate hybrid tetramers that will allow us to isolate pairwise interactions between one active site and one allosteric site. This approach will be used to answer if the allosteric site in one subunit regulates the active site in the same subunit, a result that will simplify mechanistic interpretation of structural changes. Detailed understanding of the molecular mechanisms of hL-PYK will facilitate future drug designs to activate (or prevent inhibition of) this enzyme to reduced hepatic glucose secretion and counteract hyperglycemia.
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2017 — 2020 |
Fenton, Aron W Lamb, Audrey L (co-PI) [⬀] Smith, Paul E (co-PI) [⬀] Smith, Paul E (co-PI) [⬀] Smith, Paul E (co-PI) [⬀] Smith, Paul E (co-PI) [⬀] Swint-Kruse, Liskin |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Towards Exome Analyses: Surprising Outcomes From Mutating Nonconserved Positions @ University of Kansas Medical Center
When genomes are sequenced for personalized medicine, each patient can have up to 10,000 varia- tions in their protein sequences. To identify which amino acid changes are medically relevant, many computer algorithms have been developed. In thinking about how to improve these algorithms, we considered that, among their input, many include evolutionary information about the affected proteins. Algorithms also include ?rules? devised from decades of mutation experiments: Similar amino acids allow function (toggle on); other amino acids abolish function or structure (toggle off); each mutation will have the same outcome in any homolog. However, experiments have been very heavily biased to conserved positions. In contrast, >50% of amino acid positions are not conserved during the evolution of most proteins. If nonconserved positions follow different rules, this may be one source for false positive and negative predictions in genome analyses. We are bridging this gap between experimental protein chemistry and computer predictions. In our first study, we used 10 homologs to assess the outcomes for >1000 mutations at nonconserved positions. Strikingly, these positions did not follow any of the substitution rules listed above. First, when multiple amino acids were substituted into one position, they caused a wide range of functional outcomes (?rheostat position?). Second, chemically similar amino acids did not always have similar outcomes. Third, when a given position was substituted in multiple homologs, the same amino acid had different outcomes. Thus, rheostatic nonconserved positions are likely to give false results in current predictions. Preliminary results show that other proteins have rheostat positions. The central hypothesis of this proposal is that rheostat positions have general properties that distinguish them from other nonconserved positions. In Aim 1, we will test the hypothe- sis that rheostat positions can be detected by a particular pattern of evolutionary change, using pyruvate kinase, aldolase, and an organic anion transmembrane transporter as model systems. If prediction is possible, amino acid variants at rheostat positions should be ? for now ? classified as having ?unknown significance? to reduce false predictions. Further, all experimental results can be used by the CAGI community to assess the development of new algorithms. In Aim 2, we will use molecular dynamics simulations and hydrogen ex- change experiments to determine how rheostat mutations affect protein motions. In Aim 3, we will use X-ray crystallography and structural predictions to determine how rheostat mutations affect side-chain packing. The results from Aims 2-3 (i) can be used to identify regions in other proteins that contain rheostat positions, and (ii) will provide the groundwork for formulating new rules for predicting the outcomes of rheostat mutations. The new rules are needed to reach our long-term goals of improving computer predictions and reducing the number of clinical variants with unknown significance.
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