title: Burst analysis spectroscopy: a versatile single-particle approach for studying distributions of protein aggregates and fluorescent assemblies.
abstract: Many essential cellular functions depend on the assembly and disassembly of macromolecular complexes. The size, form, and distribution of these assemblies can be heterogeneous and complex, rendering their detailed characterization difficult. Here we describe a simple non-correlation-based method capable of directly measuring population distributions at very low sample concentrations. Specifically, we exploit the highest signal-to-noise light bursts from single fluorescent particles transiting a confocal excitation spot to recursively determine the brightness and size distribution of complex mixtures of fluorescent objects. We refer to this method as burst analysis spectroscopy (BAS) and demonstrate the sensitivity of this technique by examining the free-solution, time-resolved distribution of assembled protein aggregates by using two fluorescently labeled proteins: the aggregation-prone, chaperonin-dependent, folding model protein ribulose-bisphosphate carboxylase/oxygenase (RuBisCO), and an amyloidogenic fragment of the yeast prion protein Sup35. We find that the assembly kinetics of both proteins display complex multimodal behavior not readily quantifiable with other methods.
authors.affiliation: Department of Physics, Princeton University, Princeton, NJ 08544, USA. puchalla@princeton.edu
authors.forename: Jason
authors.initials: J
authors.lastname: Puchalla
authors.forename: Kelly
authors.initials: K
authors.lastname: Krantz
authors.forename: Robert
authors.initials: R
authors.lastname: Austin
authors.forename: Hays
authors.initials: H
authors.lastname: Rye
mesh_terms: D005453:Fluorescence; D007700:Kinetics; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism*; D010454:Peptide Termination Factors; D011328:Prions / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D046912:Multiprotein Complexes; D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012273:Ribulose-Bisphosphate Carboxylase
references.citation: Nat Methods. 2006 Feb;3(2):83-9
references.pmid: 16432516
references.citation: J Biotechnol. 2001 Apr 13;86(3):163-80
references.pmid: 11257530
references.citation: Biophys J. 1988 Dec;54(6):983-93
references.pmid: 3233275
references.citation: Biophys J. 1999 Jul;77(1):553-67
references.pmid: 10388780
references.citation: Nat Biotechnol. 2006 Mar;24(3):358-62
references.pmid: 16474383
references.citation: Annu Rev Neurosci. 2003;26:267-98
references.pmid: 12704221
references.citation: PLoS Biol. 2004 Oct;2(10):e321
references.pmid: 15383837
references.citation: Proc Natl Acad Sci U S A. 2000 May 9;97(10):5468-73
references.pmid: 10805803
references.citation: Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15492-7
references.pmid: 14673112
references.citation: Annu Rev Biochem. 2006;75:333-66
references.pmid: 16756495
references.citation: Biochem Biophys Res Commun. 2005 Aug 12;333(4):1202-10
references.pmid: 15978545
references.citation: Biophys J. 1996 Jul;71(1):410-20
references.pmid: 8804624
references.citation: Appl Opt. 1991 Apr 1;30(10):1185-95
references.pmid: 20582127
references.citation: Nature. 1989 Dec 21-28;342(6252):884-9
references.pmid: 10532860
references.citation: Biophys J. 2000 Jan;78(1):474-86
references.pmid: 10620311
references.citation: Biophys J. 2001 Sep;81(3):1805-22
references.pmid: 11509390
references.citation: Science. 2004 Jun 18;304(5678):1793-7
references.pmid: 15155912
references.citation: Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):13756-61
references.pmid: 10570145
references.citation: Biophys J. 2002 Oct;83(4):2300-17
references.pmid: 12324447
references.citation: AAPS J. 2006 Sep 15;8(3):E572-9
references.pmid: 17025275
references.citation: Mol Cell. 2004 Oct 8;16(1):23-34
references.pmid: 15469819
references.citation: Biophys J. 2006 Jul 1;91(1):42-54
references.pmid: 16565055
references.citation: AAPS J. 2006 Sep 08;8(3):E564-71
references.pmid: 17025274
references.citation: Methods Enzymol. 2006;413:91-102
references.pmid: 17046392
references.citation: Anal Biochem. 1989 Mar;177(2):244-9
references.pmid: 2729542
references.citation: Science. 1996 Aug 2;273(5275):622-6
references.pmid: 8662547
references.citation: Biophys J. 1997 Apr;72(4):1878-86
references.pmid: 9083691