A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously.

Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary two-color smFRET experiments can provide only limited insight into the function of biological systems, which commonly consist of more than two components. A complete understanding of complex multicomponent biological systems requires correlated information on conformational rearrangements on the one hand and transient interactions with binding partners on the other. Multicolor smFRET experiments enable the direct observation of such correlated dynamics and interactions. Here we demonstrate the power and limitations of multicolor smFRET experiments including the description of a multicolor smFRET setup and data analysis. A general analytical procedure for multicolor smFRET data is presented and applied to the multicomponent heat shock protein 90 system. This allows us to identify microscopic states in transient complexes. Conformational dynamics and nucleotide binding are simultaneously detected, which is impossible using two-color smFRET. Additionally, their correlation is quantified using 3D ensemble hidden Markov analysis, in and out of equilibrium. This method is perfectly suited for protein systems that are much more sophisticated than previously studied DNA-based systems. By extending the application to biologically relevant systems, multicolor smFRET comes of age and provides a unique mechanistic insight into protein machines.

Effects of high-fat diet, angiotensinogen (agt) gene inactivation, and targeted expression to adipose tissue on lipid metabolism and renal gene expression.

To address the role of angiotensinogen (agt) in lipid metabolism and its potential endocrine effects in vivo, we studied the effects of high-fat diet (HFD) on adult, 28-week-old agt knockout (KO) mice compared to wild type (WT) mice. Recent studies (Massiera et al., 2001) have demonstrated that reexpression of agt in adipose tissue of KO mice normalized adiposity, blood pressure, and kidney abnormalities. We therefore used microarray analysis to investigate changes in gene expression profile in kidneys of KO vs. Tg-KO mice, where agt expression is restricted to adipose tissue. Body weight, adiposity and insulin levels were significantly decreased (p < 0.05) in KO mice on a chow diet (CD) compared to WT mice, while circulating leptin levels were similar. On a high-fat diet, KO mice exhibited significantly lower bodyweight (p < 0.05), adiposity (p < 0.05), leptin, and insulin levels (p < 0.05) compared to WT mice. In agreement with previously reported changes in kidney histology, agt KO mice displayed altered expressions of genes involved in blood pressure regulation and renal function, but these levels were corrected by reexpression of agt in adipose tissue. Collectively, these findings further document important endocrine roles of adipocyte agt, in part via regulation of lipid metabolism and kidney homeostasis.