Information about unstable intermediates and transition states (TS) is needed to determine mechanisms of biopolymer self-assembly and function, but no general method has been available.
Aim 1 of this proposal tests the hypothesis that solutes are effective probes of changes in water accessible surface area (ASA) in conformational changes and interface formation in TS and intermediates. To use solutes as probes, their chemical (preferential) interactions with functional groups of biopolymers must be determined. These determinations allow solute interactions and solute effects to be interpreted and/or predicted in terms of ASA, and allow quantitative analysis of chemical and excluded volume effects of large solutes (Aim 2). Building on the recent demonstration that denaturant kinetic m-values for protein folding can be interpreted to quantify the preferential burial of amid ASA in folding to TS and hence distinguish between mechanisms, kinetic m-values for the effects of stabilizing and destabilizing solutes on rate constants for lac repressor-operator and RNA polymerase (RNAP)-promoter interactions will be determined and interpreted. Methods include rapid mix-quench kinetic studies at nM concentrations with 32P-labeled DNA and the filter binding assay. Results to date demonstrate the potential of this approach to determine the steps with large ASA changes in the RNAP mechanism, and to distinguish between models for the lac repression TS. Roles of proposed [solute]-sensing elements on the osmolyte transporter ProP and on RNAP in activating ProP and in buffering RNAP-promoter open complexes in response to osmotic stress will also be investigated. Proven methods (vapor pressure osmometry, solubility) are proposed to quantify preferential interactions of additional small, biochemically-significant solutes (including human, bacterial osmolytes) with model compounds displaying the functional groups of biopolymers. Novel dissections of these data into additive contributions from individual functional groups will be tested and applied to quantify interactions of these solutes with nucleic acid and protein groups. This information is needed to use these solutes as probes. Results to date validate additivity and demonstrate the feasibility and significance of this approach. Thermodynamic m-values quantifying effects of the widely-used series from ethylene glycol to polyethylene glycol (PEG) and other polymers on selected biopolymer processes will be determined and dissected into contributions of chemical interactions and excluded volume effects, using chemical interaction data obtained from studies with small oligomers. Contributions of changes in molecularity and changes in shape to the excluded volume component of the PEG effect will be compared with theoretical predictions. Spectroscopic studies of effects of PEG and other polymers on selected model processes (melting of oligomeric DNA or RNA triplexes, duplexes or hairpins, unfolding of cro repressor dimer and monomer variants), binding of nucleoid-associated protein IHF to its H'DNA site) are proposed.

Public Health Relevance

A wealth of structural information is now available about biopolymers and their complexes, but information about unstable intermediates and transition states is also needed to determine mechanisms of self-assembly and function of these complexes. We propose to apply the substantial information now available about the interactions of solutes with functional groups on the surfaces buried or exposed in these processes and use solute as probes of mechanisms of protein folding, transporter activation, lac repression and of formation and stabilization of the open complex in transcription initiation. The understanding of these and other noncovalent mechanisms will allow them to be regulated or inhibited in analogous ways to the regulation or inhibition of enzyme catalytic mechanisms by cofactors or drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM047022-21A1
Application #
8760637
Study Section
Macromolecular Structure and Function D Study Section (MSFD)
Program Officer
Preusch, Peter
Project Start
1992-08-01
Project End
2018-06-30
Budget Start
2014-08-01
Budget End
2015-06-30
Support Year
21
Fiscal Year
2014
Total Cost
$314,377
Indirect Cost
$105,034
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Diehl, Roger C; Guinn, Emily J; Capp, Michael W et al. (2013) Quantifying additive interactions of the osmolyte proline with individual functional groups of proteins: comparisons with urea and glycine betaine, interpretation of m-values. Biochemistry 52:5997-6010
Record Jr, M Thomas; Guinn, Emily; Pegram, Laurel et al. (2013) Introductory lecture: interpreting and predicting Hofmeister salt ion and solute effects on biopolymer and model processes using the solute partitioning model. Faraday Discuss 160:9-44; discussion 103-20
Guinn, Emily J; Kontur, Wayne S; Tsodikov, Oleg V et al. (2013) Probing the protein-folding mechanism using denaturant and temperature effects on rate constants. Proc Natl Acad Sci U S A 110:16784-9
Guinn, Emily J; Schwinefus, Jeffrey J; Cha, Hyo Keun et al. (2013) Correction to "quantifying functional group interactions that determine urea effects on nucleic Acid helix formation". J Am Chem Soc 135:9220
Guinn, Emily J; Schwinefus, Jeffrey J; Cha, Hyo Keun et al. (2013) Quantifying functional group interactions that determine urea effects on nucleic acid helix formation. J Am Chem Soc 135:5828-38
Guinn, Emily J; Pegram, Laurel M; Capp, Michael W et al. (2011) Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer. Proc Natl Acad Sci U S A 108:16932-7
Knowles, D B; LaCroix, Andrew S; Deines, Nickolas F et al. (2011) Separation of preferential interaction and excluded volume effects on DNA duplex and hairpin stability. Proc Natl Acad Sci U S A 108:12699-704
Pegram, Laurel M; Wendorff, Timothy; Erdmann, Robert et al. (2010) Why Hofmeister effects of many salts favor protein folding but not DNA helix formation. Proc Natl Acad Sci U S A 107:7716-21
Kontur, Wayne S; Capp, Michael W; Gries, Theodore J et al. (2010) Probing DNA binding, DNA opening, and assembly of a downstream clamp/jaw in Escherichia coli RNA polymerase-lambdaP(R) promoter complexes using salt and the physiological anion glutamate. Biochemistry 49:4361-73
Ma, Liang; Pegram, Laurel; Record Jr, M T et al. (2010) Preferential interactions between small solutes and the protein backbone: a computational analysis. Biochemistry 49:1954-62

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