title: Crowding effects on the small, fast-folding protein lambda6-85.
abstract: The microsecond folder lambda6-85 is a small (9.2 kDa = 9200 amu) five helix bundle protein. We investigated the stability of lambda6-85 in two different low-fluorescence crowding matrices: the large 70 kDa carbohydrate Ficoll 70, and the small 14 kDa thermophilic protein SubL. The same thermal stability of secondary structure was measured by circular dichroism in aqueous buffer, and at a crowding fraction phi = 15 +/- 1% of Ficoll 70. Tryptophan fluorescence detection (probing a tertiary contact) yielded the same thermal stability in Ficoll, but 4 degrees C lower in aqueous buffer. Temperature-jump kinetics revealed that the relaxation rate, corrected for bulk viscosity, was very similar in Ficoll and in aqueous buffer. Thus viscosity, hydrodynamics and crowding seem to compensate one another. However, a new fast phase was observed in Ficoll, attributed to crowding-induced downhill folding. We also measured the stability of lambda6-85 in phi = 14 +/- 1% SubL, which acts as a smaller more rigid crowder. Significantly greater stabilization (7 to 13 degrees C depending on probe) was observed than in the Ficoll matrix. The results highlight the importance of crowding agent choice for studies of small, fast-folding proteins amenable to comparison with molecular dynamics simulations.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois, 600 South Mathews Avenue, Urbana-Champaign, IL 61801, USA.
authors.forename: Sharlene
authors.initials: S
authors.lastname: Denos
authors.forename: Apratim
authors.initials: A
authors.lastname: Dhar
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry*; D013816:Thermodynamics; D013997:Time Factors; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry*
chemical_list: D012097:Repressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins