Proteins fold under mechanical forces in a number of biological processes, ranging from muscle contraction to co-translational folding. As force hinders the folding transition, chaperones must play a role in this scenario, although their influence on protein folding under force has not been directly monitored yet. Here, we introduce single-molecule magnetic tweezers to study the folding dynamics of protein L in presence of the prototypical molecular chaperone trigger factor over the range of physiological forces (4–10 pN). Our results show that trigger factor increases prominently the probability of folding against force and accelerates the refolding kinetics. Moreover, we find that trigger factor catalyzes the folding reaction in a force-dependent manner; as the force increases, higher concentrations of trigger factor are needed to rescue folding. We propose that chaperones such as trigger factor can work as foldases under force, a mechanism which could be of relevance for several physiological processes.

Here, we study the human serum albumin (HSA) protein–Au nanoparticle interaction to identify the specific binding site of protein with nanoparticles by using the surface energy transfer (SET) method among tryptophan (Trp) of HSA, ANS-dye-labeled HSA protein, and Au nanoparticles. Here, ANS dye is used as a probe located at domain IIIA of HSA. In particular, absorbance, fluorescence quenching, decay time, circular dichroism, dynamic light scattering, and TEM measurements are performed to understand the physical properties of protein-conjugated Au nanoparticles. Using the SET method, the measured distances between the Trp residue of HSA and the binding site of HSA interacting with Au nanoparticles are 42.5, 41.9, and 48.1 Å for 1.5, 2.0, and 2.9 nm HSA-conjugated Au nanoparticles, respectively. The measured distances between the binding site of ANS dye (located at domain IIIA) in HSA to the binding site of HSA interacting with Au nanoparticles are 51, 51.5, and 54.7 Å for 1.5, 2.0, and 2.9 nm HSA-conjugated Au nanoparticles, respectively. From the protein structural data (using PyMol software), the distances from the center of domain IIIA to Cys53–Cys62 disulfide bond and Trp to Cys53–Cys62 disulfide bond are obtained to be 51.5 and 39.1 Å, respectively. Thus, the distances calculated by using SET equation (Trp to Au binding site distance and ANS to Au binding site distance) nicely match with the distances obtained from protein structural data by using PyMol software. Analysis suggests that the Au nanoparticle is attached to HSA by linkage through Cys53–Cys62 disulfide bond which is located at subdomain IA of HSA.

SUMMARY The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent molecular systems. While the mechanisms of force generation have been studied at the single molecular motor level, there is little understanding of the magnitude of power that can be generated by folding proteins. Here, we use single-molecule force spectroscopy techniques to measure the force-velocity relation of folding titin domains that contain single internal disulfide bonds, a common feature throughout the titin I-band. We find that formation of the disulfide regulates the peak power output of protein folding in an all-or-none manner, providing at 6.0 pN, for example, a boost from 0 to 6,000 zW upon oxidation. This mechanism of power generation from protein folding is of great importance for muscle, where titin domains may unfold and refold with each extension and contraction of the sarcomere.

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r H ) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r H of 29 Å ) over an extended one (r H of 40 Å ), which forms in their absence. Also, the time constant of a kinetic component ( R ) of about 30 s has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.