Fundamental link between folding states and functional states of proteins.
Folding and function of proteins are two aspects of proteins which are usually considered as basically unrelated phenomena that are optimized by evolution independently. From the funnel model of folding/unfolding and the associated energy landscape, we infer the paradigm that the minimum number of folding intermediates is determined by the number of all functional states of a protein ("essential" folding intermediates). Here, we demonstrate the supposed fundamental link using the Ras protein complexed with the GTP analogue GppNHp that occurs in two structural states coexisting in solution. State 2 was shown earlier to represent the effector interacting state, and the function of state 1 was hitherto unknown. By (31)P NMR spectroscopy, we demonstrate that state 1 represents the conformation interacting with guanine nucleotide exchange factors (GEFs). Denaturation experiments of the protein with a chaotropic reagent show that both functional states coexist during folding and unfolding. Application of high pressure represents another perturbation of the energy landscape, leading to an increased population of the state 1 as observed by NMR spectroscopy. The specific volume difference between the two states DeltaV(12) is 17.2 +/- 0.5 mL mol(-1), indicating that state 1 represents a more open conformation of the protein. The free energies of stabilization for state 1 and state 2 at 278 K can be determined as 8.3 and 9.8 kJ mol(-1), respectively.
Institute of Biophysics and Physical Biochemistry, University of Regensburg, D-93040 Regensburg, Germany. hans-robert.kalbitzer@biologie.uni-r.de
Hans Robert
HR
Kalbitzer
Michael
M
Spoerner
Petra
P
Ganser
Constantin
C
Hozsa
Werner
W
Kremer
D019277:Entropy; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D015398:Signal Transduction; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate; D018631:ras Proteins