title: Ligand binding modulates the mechanical stability of dihydrofolate reductase.
abstract: We use single-molecule force spectroscopy to demonstrate that the mechanical stability of the enzyme dihydrofolate reductase (DHFR) is modulated by ligand binding. In the absence of bound ligands, DHFR extends at very low forces, averaging 27 pN, without any characteristic mechanical fingerprint. By contrast, in the presence of micromolar concentrations of the ligands methotrexate, nicotinamide adenine dihydrogen phosphate, or dihydrofolate, much higher forces are required (82 +/- 18 pN, 98 +/- 15 pN, and 83 +/- 16 pN, respectively) and a characteristic fingerprint is observed in the force-extension curves. The increased mechanical stability triggered by these ligands is not additive. Our results explain the large reduction in the degradation rate of DHFR, in the presence of its ligands. Our observations support the view that the rate-limiting step in protein degradation by adenosine triphosphate-dependent proteases is the mechanical unfolding of the target protein.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY, USA.
authors.forename: Sri Rama Koti
authors.initials: SR
authors.lastname: Ainavarapu
authors.forename: Lewyn
authors.initials: L
authors.lastname: Li
authors.forename: Carmen L
authors.initials: CL
authors.lastname: Badilla
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernandez
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008727:Methotrexate / Q000494:pharmacology; D018625:Microscopy, Atomic Force; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D009536:Niacinamide / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D021381:Protein Transport; D013053:Spectrophotometry; D013314:Stress, Mechanical; D013718:Tensile Strength; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D005493:Folic Acid Antagonists; D008024:Ligands; D009243:NAD; D009536:Niacinamide; C010920:dihydrofolate; D009249:NADP; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D046988:Proteasome Endopeptidase Complex; D008727:Methotrexate