Nonideality and protein thermal denaturation

Waldner, Jennifer C.; Lahr, Steven J.; Edgell, Marshall H.; Pielak, Gary J.

doi:10.1002/(sici)1097-0282(199905)49:6<471::aid-bip5>3.0.co;2-z

Cited by 17 publications

(10 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…27,33 An increased stability against denaturation in highly occupied volume of homogeneous solutions is reported also in Ref. 35 All these experimental observations support our theoretical predictions.…”

Section: Comparison With Experimentssupporting

confidence: 87%

“…An enhancement of the protein stability at high protein concentration was reported in experimental measurements. 35 We can predict also the level of self-stabilization expected during unfolding of adenylate kinase for a f = 20% volume occupancy in solution. The self-stabilization occurs due to steric effects induced by the unfolding of a fraction of proteins in solution.…”

Section: Resultsmentioning

confidence: 99%

“…35 The radius of gyration r g of a protein (the radial size which defines how compact the protein structure is) expands during unfolding excluding volume to other surrounding proteins. If the increase r of the radius of gyration is in the range of the protein interspace, defined as the mean distance between proteins in solution [d = (3/4 π n) −1/3 , where n is the protein concentration], the subsequent confinement would provide stability for adjacent proteins which are in compact, native states.…”

Section: Effects Of Crowding On the Unfolding/folding Kinetic Ratesmentioning

confidence: 99%

See 2 more Smart Citations

Effects of Crowding on the Thermal Stability of Heterogeneous Protein Solutions

2005

View full text Add to dashboard Cite

Crowding can substantially affect the transition of a protein between its native (N) and unfolded (U) states via volume exclusion effects. Also, it influences considerably the aggregation (A) of unfolded proteins. To examine the details, we developed an approach for computing the kinetic rates of the process N <--> U --> A in which the concentration of the protein is explicitly taken into account. We then compute the relative change with temperature of the protein denaturation for various fractional volume occupancies and partition of proteins in solution. The analysis indicates that, in protein solutions in which the average distance between proteins is comparable with the radius of gyration of an unfolded protein, steric effects increase the stability of the proteins which are in compact, native states. In heterogeneous protein solutions containing various types of proteins with different thermal stabilities, the unfolding of the most thermolabile proteins will increase the stability of the other proteins. The results shed light on the way proteins change the thermal stability of a cell as they unfold and aggregate. This study may be valuable in questions related to the dynamics of thermal injuries.

show abstract

Section: Comparison With Experimentssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Effects Of Crowding On the Unfolding/folding Kinetic Ratesmentioning

confidence: 99%

See 1 more Smart Citation

Effects of Crowding on the Thermal Stability of Heterogeneous Protein Solutions

2005

View full text Add to dashboard Cite

show abstract

“…We chose eglin c as our model protein because it is a small 70-residue protein with both an ␣-helix and a ␤-sheet and no cysteines. Its structure is known both from NMR (25) and x-ray crystallography (26), its denaturation thermodynamics is well defined (27,28), and its structural homologue, CI-2, has proven to be a useful model protein for folding (29)(30)(31) and mutagenesis studies (32,33). We also wanted a protein with a straightforward activity assay so that we could use high-throughput methods to monitor the consequences of mutation.…”

Section: Resultsmentioning

confidence: 99%

Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

Lahr

Broadwater

Carter

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an ␣-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R 2 ‫؍‬ 0.97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques. Site-directed mutagenesis (1) and combinatorial libraries (2-19) have been used to generate considerable information about the structure-determining elements in proteins. The fraction of variants in combinatorial libraries containing randomized residues (2-8) or residues constrained to be hydrophobic (9-15) that pass some test have been used as semiquantitative assessments of the role of the targeted residues or motifs. A more hypothesis-oriented approach used degenerate codons to construct binary or hydrophobic-hydrophilic patterns in library variants whose effects on protein structure could then be tested (16)(17)(18)(19). However, only a limited number of types of hypotheses can be tested if one uses degenerate codons (codons with mixed nucleotides at one or more sites) to introduce variability into a library. We report here two tools to extend the range of hypotheses that can be quantitatively assessed with combinatorial libraries.The first extension involves the use of resin-splitting technology (20) to facilitate the construction of arbitrarily complex libraries that are free of the constraints imposed by the genetic code. Libraries can be constructed so that all of their members conform to some hypothesis, and members can then be scored by some structurally related test. As in previous applications, the successful fraction of variants serves as a relative ''score'' of the hypothesis, but the arbitrarily complex nature of the hypotheses made possible by split-resin technology extends the range of what can be tested.The second extension involves the use of regression analysis to expand the analytical power of combinatorial library experiments. In an ideal case, one would be able to assess a hypothesis concerning the determinants of protein structure by making a small number of carefully chosen mu...

show abstract

“…We chose eglin c as our model protein because it is a small, 70 residue, protein with both an a-helix and a ß-sheet and no cysteines. Its structure is known both from NMR (35) and x-ray crystallography (36), its denaturation thermodynamics is well defined (37,38) and its structural homologue, CI-2, has proven to be a wellmannered protein in folding (39)(40)(41)(42)(43) and mutagenesis studies (44)(45)(46)(47). We also wanted a protein with a straight-forward activity assay so that we could use high-throughput methods to monitor the consequences of mutation.…”

Section: Resultsmentioning

confidence: 99%

A New Generic Method for the Production of Protein-Based Inhibitors of Proteins Involved in Cancer Metastasis

Edgell¹

1999

View full text Add to dashboard Cite

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this' collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 121B Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project 10704-0188), Washington, DC 20503 AGENCY USE ONLY (Leave blank)2. REPORT DATE August 1999 REPORT TYPE AND DATES COVEREDFinal (lAug 94-31 Jul 99) TITLE AND SUBTITLE A New Generic Method for the Production of Protein-Based Inhibitors of Proteins Involved in Cancer Metastasis AUTHOR(S)Marshall H. Edgell, Ph.D. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)University of North Carolina, Chapel Hill Chapel Hill, North Carolina 27599-4100 Our objective was to develop a method to make protein-based inhibitors against protein targets and as the test case to a proteinase involved in metastasis, stromelysin. The approach used phage display and a display framework which was expected to bind preferentially to the active site pocket of target proteins. While we were able to find peptides in phage display libraries that bound to active stromelysin or to cadmium inactivated stromelysin and could find binders to a test target, papain, in a constrained loop library we were unable to get the fully constrained scaffold protein to bind to its cognate target, subtilisin in the phage display system. Hence the overall objective was not obtained. As part of our efforts to characterize the binding epitope display framework, eglin c, we did develop a new method for using mutagenesis to study protein structure which we call patterned library analysis. We carried out a proof-of-principle experiment using the new method in which we were able to reproduce the values for cc-helix propensity indicating that the method does indeed work. This method should have broad utility as a method for the assessment of hypotheses concerning the determinants of protein structure. Where copyrighted material is quoted, permission has been obtained to use such material. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)U SUBJECT TERMSWhere material from documents designated for limited distribution is quoted, permission has been obtained to use the material.Citations of commercial organizations and trade names in this report do not constitute an official Department of Army endorsement or approval of the products or services of these organizations.mA&T In conducting research using animals, the investigator(s) adhered to the "Guide for the Care and Use of Laboratory Animals," prepared by the Committee on Care and use of Laboratory Animals of the Institute of Laboratory Resources, national Research Council ...

show abstract

Nonideality and protein thermal denaturation

Cited by 17 publications

References 40 publications

Effects of Crowding on the Thermal Stability of Heterogeneous Protein Solutions

Effects of Crowding on the Thermal Stability of Heterogeneous Protein Solutions

Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

A New Generic Method for the Production of Protein-Based Inhibitors of Proteins Involved in Cancer Metastasis

Contact Info

Product

Resources

About