How to scan naked DNA using promiscuous recognition and no clamping: a model for pioneer transcription factors.

Most DNA scanning proteins uniquely recognize their cognate sequence motif and slide on DNA assisted by some sort of clamping interface. The pioneer transcription factors that control cell fate in eukaryotes must forgo both elements to gain access to DNA in naked and chromatin forms; thus, whether or how these factors scan naked DNA is unknown. Here, we use single-molecule techniques to investigate naked DNA scanning by the Engrailed homeodomain (enHD) as paradigm of highly promiscuous recognition and open DNA binding interface. We find that enHD scans naked DNA quite effectively, and about 200000-fold faster than expected for a continuous promiscuous slide. To do so, enHD scans about 675 bp of DNA in 100 ms and then redeploys stochastically to another location 530 bp afar in just 10 ms. During the scanning phase enHD alternates between slow- and medium-paced modes every 3 and 40 ms, respectively. We also find that enHD binds nucleosomes and does so with enhanced affinity relative to naked DNA. Our results demonstrate that pioneer-like transcription factors can in principle do both, target nucleosomes and scan active DNA efficiently. The hybrid scanning mechanism used by enHD appears particularly well suited for the highly complex genomic signals of eukaryotic cells.

Hypertrophic cardiomyopathy mutations Y115H and E497D disrupt the folded-back state of human beta-cardiac myosin allosterically.

At the molecular level, clinical hypercontractility associated with many hypertrophic cardiomyopathy (HCM)-causing mutations in beta-cardiac myosin appears to be driven by their disruptive effect on the energy-conserving, folded-back, super relaxed (SRX) OFF-state of myosin. A pathological increase in force production results from release of heads from this OFF-state, which results in an increase in the number of heads free to interact with actin and produce force. Pathogenic mutations in myosin can conceivably disrupt the OFF-state by (1) directly affecting the intramolecular interfaces stabilizing the folded-back state, or (2) allosterically destabilizing the folded-back state via disruption of diverse conformational states of the myosin motor along its chemomechanical cycle. However, very little is understood about the mutations that fall in the latter group. Here, using recombinant human beta-cardiac myosin, we analysed the biomechanical properties of two such HCM-causing mutations, Y115H (in the transducer) and E497D (in the relay helix), neither of which falls in the regions that interact to stabilize the myosin folded-back state. We find these mutations have diverse effects on the contractility parameters of myosin, yet the primary hypercontractile change in both cases is the destabilization of the OFF-state of myosin and increased availability of active myosin heads for actin-binding. Experimental data and molecular dynamics simulations indicate that these mutations likely destabilize the pre-powerstroke state of myosin, the conformation the motor adopts in the inactive folded-back state. We propose that destabilization of the folded-back state of myosin, directly and/or allosterically, is the molecular basis of hypercontractility in HCM in a far greater number of pathogenic mutations than currently thought.

Microsecond sub-domain motions and the folding and misfolding of the mouse prion protein.

Protein aggregation appears to originate from partially unfolded conformations that are sampled through stochastic fluctuations of the native protein. It has been a challenge to characterize these fluctuations, under native like conditions. Here, the conformational dynamics of the full-length (23-231) mouse prion protein were studied under native conditions, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS). The slowest fluctuations could be associated with the folding of the unfolded state to an intermediate state, by the use of microsecond mixing experiments. The two faster fluctuations observed by PET-FCS, could be attributed to fluctuations within the native state ensemble. The addition of salt, which is known to initiate the aggregation of the protein, resulted in an enhancement in the time scale of fluctuations in the core of the protein. The results indicate the importance of native state dynamics in initiating the aggregation of proteins.

Ruggedness in the Free Energy Landscape Dictates Misfolding of the Prion Protein.

Experimental determination of the key features of the free energy landscapes of proteins, which dictate their adeptness to fold correctly, or propensity to misfold and aggregate and which are modulated upon a change from physiological to aggregation-prone conditions, is a difficult challenge. In this study, sub-millisecond kinetic measurements of the folding and unfolding of the mouse prion protein reveal how the free energy landscape becomes more complex upon a shift from physiological (pH 7) to aggregation-prone (pH 4) conditions. Folding and unfolding utilize the same single pathway at pH 7, but at pH 4, folding occurs on a pathway distinct from the unfolding pathway. Moreover, the kinetics of both folding and unfolding at pH 4 depend not only on the final conditions but also on the conditions under which the processes are initiated. Unfolding can be made to switch to occur on the folding pathway by varying the initial conditions. Folding and unfolding pathways appear to occupy different regions of the free energy landscape, which are separated by large free energy barriers that change with a change in the initial conditions. These barriers direct unfolding of the native protein to proceed via an aggregation-prone intermediate previously identified to initiate the misfolding of the mouse prion protein at low pH, thus identifying a plausible mechanism by which the ruggedness of the free energy landscape of a protein may modulate its aggregation propensity.

A Dry Transition State More Compact Than the Native State Is Stabilized by Non-Native Interactions during the Unfolding of a Small Protein.

Defining the role of non-native interactions in directing the course of protein folding or unfolding reactions has been a difficult challenge. In particular, the extent to which such interactions play a productive role by stabilizing the structures of transition states (TSs) found on the folding and unfolding pathways of proteins is not known. On the contrary, it is thought that the TSs are expanded forms of the N state stabilized by native interactions, and it is not known whether non-native interactions can modulate TS structure. In this study of the unfolding of the SH3 domain of PI3 kinase using a microsecond mixing methodology, partial non-native structure formation is shown to occur initially during unfolding. The TS of this partial "folding during unfolding" reaction is more compact than the N state: the apparent rate constant of Trp53 burial during this reaction decreases with an increase in denaturant concentration. Kinetic studies of the unfolding of mutant variants suggest that the unusually compact TS is stabilized by interactions not present in N and that these non-native interactions are hydrophobic in nature. It was determined that mutation could be used to tune the degree of compaction in the TS.

Microsecond Rearrangements of Hydrophobic Clusters in an Initially Collapsed Globule Prime Structure Formation during the Folding of a Small Protein.

Determining how polypeptide chain collapse initiates structure formation during protein folding is a long standing goal. It has been challenging to characterize experimentally the dynamics of the polypeptide chain, which lead to the formation of a compact kinetic molten globule (MG) in about a millisecond. In this study, the sub-millisecond events that occur early during the folding of monellin from the guanidine hydrochloride-unfolded state have been characterized using multiple fluorescence and fluorescence resonance energy transfer probes. The kinetic MG is shown to form in a noncooperative manner from the unfolded (U) state as a result of at least three different processes happening during the first millisecond of folding. Initial chain compaction completes within the first 37µs, and further compaction occurs only after structure formation commences at a few milliseconds of folding. The transient nonnative and native-like hydrophobic clusters with side chains of certain residues buried form during the initial chain collapse and the nonnative clusters quickly disassemble. Subsequently, partial chain desolvation occurs, leading to the formation of a kinetic MG. The initial chain compaction and subsequent chain rearrangement appear to be barrierless processes. The two structural rearrangements within the collapsed globule appear to prime the protein for the actual folding transition.

Rise of the Helix from a Collapsed Globule during the Folding of Monellin.

Early kinetic intermediates observed during the folding of many proteins are invariably compact and appear to possess some secondary structure. Consequently, it has been difficult to understand whether compaction drives secondary structure formation or secondary structure formation facilitates compaction during folding. In this study of the folding of single-chain monellin, it is shown that a kinetic molten globule (MG) is populated at 2 ms of folding. Far-UV circular dichroism (CD) measurements show that the kinetic MG is devoid of any helical structure even under the most stabilizing folding conditions. Multisite fluorescence resonance energy transfer (FRET) measurements show that the kinetic MG is compact with different segments having contracted to different extents. It is shown that the sequence segment that goes on to form the sole helix in the native protein is fully collapsed in the kinetic MG. This segment expands to accommodate the helix as the kinetic MG folds further to the native state, while other segments of the protein contract. Helix formation starting from the kinetic MG is shown to occur in multiple kinetic steps, whether measured by far-UV CD or by FRET.