Molecular chaperones are known to be essential for avoiding protein aggregation in vivo, but it is still unclear how they affect protein folding mechanisms. We use single-molecule Förster resonance energy transfer to follow the folding of a protein inside the GroEL/GroES chaperonin cavity over a time range from milliseconds to hours. Our results show that confinement in the chaperonin decelerates the folding of the C-terminal domain in the substrate protein rhodanese, but leaves the folding rate of the N-terminal domain unaffected. Microfluidic mixing experiments indicate that strong interactions of the substrate with the cavity walls impede the folding process, but the folding hierarchy is preserved. Our results imply that no universal chaperonin mechanism exists. Rather, a competition between intra-and intermolecular interactions determines the folding rates and mechanisms of a substrate inside the GroEL/GroES cage.I n the recent past, a large number of components have been identified that control and modulate protein folding in vivo. This machinery includes molecular chaperones (1-3), sophisticated quality control systems, and complex mechanisms for protein translocation and degradation (3, 4), reflecting the importance of regulating the delicate balance of protein folding, misfolding, and aggregation in the cell. Such cellular factors exert conformational constraints on protein molecules that are expected to have a strong effect on the corresponding free-energy surfaces for folding (5). However, while the combination of cellular, biochemical, and structural data has led to some plausible qualitative models for the processes involved, mechanistic investigations comparable to those of autonomous protein folding in vitro (5-8) have been complicated by the complexity of the systems and the conformational heterogeneity involved (9). Even the autonomous folding of chaperone substrate proteins has been difficult to investigate because of their strong aggregation tendency (10). Contributions from confinement and crowding have been addressed in numerous studies using molecular simulations and theory (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but many of these concepts have eluded experimental examination.Here, we take a step towards closing this gap by investigating the GroEL/GroES chaperonin (1-3, 9) with single-molecule fluorescence spectroscopy (21-24), a method that is starting to provide previously inaccessible information on chaperonemediated protein folding (25)(26)(27)(28)(29)(30). GroEL/GroES is a remarkable molecular machine that binds nonnative proteins and allows them to fold within a cavity formed by the heptameric rings of GroEL and GroES. However, the cavity is only slightly larger than the folded structure of typical proteins known to interact with the chaperonin. The large volume of unconfined unfolded protein chains compared to the size of the cavity raises the question of whether and how such strong confinement affects the folding reaction (12-16, 18, 31, 32). By labeling the classic substrate prote...

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene and has some features similar to the CC family of chemokines. was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, was also abundant in the cervix. In skin, was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from -deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis.

Molecular chaperones aid protein folding in the cell, but their effects on the conformation of the substrate protein have largely eluded experimental investigation. Single‐molecule fluorescence spectroscopy was used to extract structural and dynamic information from a protein–chaperone complex (see figure; yellow: rhodanase, blue: GroEL). This approach will aid in a more physical understanding of the role of cellular factors in protein folding.

Pro-forms of growth factors have received intensive scientific attention recently because in some cases different biological activities have been ascribed compared with the mature growth factors. Examples are the pro-apoptotic role of the nerve growth factor (NGF) proform (proNGF) or the latency of the transforming growth factor (TGF)-␤ pro-form (proTGF-␤). To investigate a possible biological function of the pro-form of bone morphogenetic protein (BMP)-2, a member of the TGF-␤ family, mature BMP-2, proBMP-2, and the isolated pro-peptide were recombinantly produced in Escherichia coli cells, and a biophysical comparison was performed. Protocols were developed that allowed efficient refolding and subsequent purification of the proteins. ProBMP-2 could be processed to an N-terminally truncated form of BMP-2, digit removed BMP-2 (drBMP-2), that possessed biological activity, i.e. it induced ectopic bone formation. Bone inducing activity was also displayed by proBMP-2. The three proteins were characterized both by fluorescence and CD spectroscopy. From these analyses, predominant ␤-sheet secondary structural elements in the pro-peptide were deduced. The thermodynamic stability of the pro-peptide was determined by chemical unfolding assays. As in the case of NGF/proNGF, the mature part of BMP-2 stabilized the structure of the pro-peptide moiety. However, in contrast to NGF/proNGF, the pro-peptide did not stimulate oxidative folding of the mature part in vitro.

Time-correlated single photon counting continues to gain importance in a wide range of applications. Most prominently, it is used for time-resolved fluorescence measurements with sensitivity down to the single molecule level. While the primary goal of the method used to be the determination of fluorescence lifetimes upon optical excitation by short light pulses, recent modifications and refinements of instrumentation and methodology allow for the recovery of much more information from the detected photons, and enable entirely new applications. This is achieved most successfully by continuously recording individually detected photons with their arrival time and detection channel information (time tagging), thus avoiding premature data reduction and concomitant loss of information. An important property of the instrumentation used is the number of detection channels and the way they interrelate. Here we present a new instrument architecture that allows scalability in terms of the number of input channels while all channels are synchronized to picoseconds of relative timing and yet operate independent of each other. This is achieved by means of a modular design with independent crystal-locked time digitizers and a central processing unit for sorting and processing of the timing data. The modules communicate through high speed serial links supporting the full throughput rate of the time digitizers. Event processing is implemented in programmable logic, permitting classical histogramming, as well as time tagging of individual photons and their temporally ordered streaming to the host computer. Based on the time-ordered event data, any algorithms and methods for the analysis of fluorescence dynamics can be implemented not only in postprocessing but also in real time. Results from recently emerging single molecule applications are presented to demonstrate the capabilities of the instrument.Scalable time-correlated photon counting system with multiple independent input channels Time-correlated single photon counting continues to gain importance in a wide range of applications. Most prominently, it is used for time-resolved fluorescence measurements with sensitivity down to the single molecule level. While the primary goal of the method used to be the determination of fluorescence lifetimes upon optical excitation by short light pulses, recent modifications and refinements of instrumentation and methodology allow for the recovery of much more information from the detected photons, and enable entirely new applications. This is achieved most successfully by continuously recording individually detected photons with their arrival time and detection channel information ͑time tagging͒, thus avoiding premature data reduction and concomitant loss of information. An important property of the instrumentation used is the number of detection channels and the way they interrelate. Here we present a new instrument architecture that allows scalability in terms of the number of input channels while all channels are synchronized ...

The misfolding and aggregation of proteins is a common phenomenon both in the cell, in in vitro protein refolding, and the corresponding biotechnological applications. Most importantly, it is involved in a wide range of diseases, including some of the most prevalent neurodegenerative disorders. However, the range of methods available to analyze this highly heterogeneous process and the resulting aggregate structures has been very limited. Here we present an approach that uses confocal single molecule detection of FRET-labeled samples employing four detection channels to obtain information about diffusivity, anisotropy, fluorescence lifetimes and Förster transfer efficiencies from a single measurement. By combining these observables, this method allows the separation of subpopulations of folded and misfolded proteins in solution with high sensitivity and a differentiation of aggregates generated under different conditions. We demonstrate the versatility of the method with experiments on rhodanese, an aggregation-prone two-domain protein.

The bacterial chaperonin GroEL/GroES assists folding of a broad spectrum of denatured and misfolded proteins. Here, we explore the limits of this remarkable promiscuity by mapping two denatured proteins with very different conformational properties, rhodanese and cyclophilin A, during binding and encapsulation by GroEL/GroES with single-molecule spectroscopy, microfluidic mixing, and ensemble kinetics. We find that both proteins bind to GroEL with high affinity in a reaction involving substantial conformational adaptation. However, whereas the compact denatured state of rhodanese is encapsulated efficiently upon addition of GroES and ATP, the more expanded and unstructured denatured cyclophilin A is not encapsulated but is expelled into solution. The origin of this surprising disparity is the weaker interactions of cyclophilin A with a transiently formed GroEL-GroES complex, which may serve as a crucial checkpoint for substrate discrimination.

Molekulare Chaperone unterstützen die Proteinfaltung in der Zelle, ihr Einfluss auf die Konformation ihrer Substratproteine war bislang aber wenig verstanden. Einzelmolekülfluoreszenzspektroskopie wurde nun eingesetzt, um Informationen über Struktur und Dynamik von Protein‐Chaperon‐Komplexen (siehe Abbildung; gelb: Rhodanese, blau: GroEL) zu erhalten. Dieser Ansatz ebnet den Weg für ein physikalisches Verständnis der Rolle zellulärer Faktoren in der Proteinfaltung.