Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity.
Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.5, 2.7, 4.2, and 5.3 M urea. CD profiles of RPA unfolding are characterized by a single transition. RPA is stabilized with respect to the CD-monitored transition when bound to a dA15 oligonucleotide. However, oligonucleotide binding appears to exert little, if any, effect on the first fluorescence transition. Methylene carbene chemical modification, coupled with MALDI-TOF mass spectrometry analysis, was also used to monitor unfolding of several specific RPA folds of the protein. The unfolding profiles of the individual structures are characterized by single transitions similar to the CD-monitored transition. Each fold, however, unravels with different individual characteristics, suggesting significant autonomy. Based on results from chemical modification and spectroscopic analyses, we conclude the initial transition observed in fluorescence experiments represents a change in the juxtaposition of binding folds with little unraveling of the domain structures. The second transition represents the unfolding of the majority of fold structure, and the third transition observed by fluorescence correlates with the dissociation of the 70- and 32-kD subunits.
Nuss
Jonathan E
JE
Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio 45435-0001, USA.
Alter
Gerald M
GM
D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006838:Hydrocarbons; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000032:analysis / Q000737:chemistry; D051716:Replication Protein A; D013057:Spectrum Analysis
D004268:DNA-Binding Proteins; D006838:Hydrocarbons; D009841:Oligonucleotides; D021122:Protein Subunits; D051716:Replication Protein A; C030011:carbene; D008697:Methane