Mega-scale experimental analysis of protein folding stability in biology and design.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

Dissecting the stability determinants of a challenging de novo protein fold using massively parallel design and experimentation.

Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet, some protein folds are easier to design than others. Previous work identified the 43-residue ɑßßɑ fold as especially challenging: The best designs had only a 2% success rate, compared to 39 to 87% success for other simple folds [G. J. Rocklin et al., Science 357, 168-175 (2017)]. This suggested the ɑßßɑ fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over 10,000 new ɑßßɑ proteins and found over 3,000 of them to fold into stable structures using a high-throughput protease-based assay. NMR, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed ɑßßɑ structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the ɑßßɑ topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

Fusion with heat-resistant obscure (Hero) proteins have the potential to improve the molecular property of recombinant proteins.

Although recombinant proteins are widely used in biotechnology and pharmaceutical industries, improving their solubility and stability is often a challenging issue. We recently discovered a class of highly unstructured heat-resistant obscure (Hero) proteins, which function to protect other "client" proteins in trans from various stresses in vitro and in vivo. Here, we show that fusion of Hero proteins in cis can enhance the molecular property of recombinant proteins. Fusion with Hero11 improved the otherwise challenging production of TAR DNA-binding protein of 43 kDa (TDP-43) in Escherichia coli. Moreover, fusing with Hero9 strongly protected the activity of firefly luciferase bearing destabilizing mutations against heat and other stress conditions. These data suggest that Hero proteins have the potential to be used as versatile stabilization tags for recombinant protein production.

A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation.

Proteins are typically denatured and aggregated by heating at near-boiling temperature. Exceptions to this principle include highly disordered and heat-resistant proteins found in extremophiles, which help these organisms tolerate extreme conditions such as drying, freezing, and high salinity. In contrast, the functions of heat-soluble proteins in non-extremophilic organisms including humans remain largely unexplored. Here, we report that heat-resistant obscure (Hero) proteins, which remain soluble after boiling at 95°C, are widespread in Drosophila and humans. Hero proteins are hydrophilic and highly charged, and function to stabilize various "client" proteins, protecting them from denaturation even under stress conditions such as heat shock, desiccation, and exposure to organic solvents. Hero proteins can also block several different types of pathological protein aggregations in cells and in Drosophila strains that model neurodegenerative diseases. Moreover, Hero proteins can extend life span of Drosophila. Our study reveals that organisms naturally use Hero proteins as molecular shields to stabilize protein functions, highlighting their biotechnological and therapeutic potential.

Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems.

Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form.

ATG conjugation-dependent degradation of the inner autophagosomal membrane is a key step for autophagosome maturation.

Although the autophagy-related (ATG) conjugation systems are thought to be important for a late step of autophagosome formation, their precise function has been poorly understood because they are also required for localization of the most important autophagosomal marker LC3. In our recent study we found that, using the autophagosomal SNARE STX17 (syntaxin 17) as an alternative marker, autophagosome-like structures were generated in ATG conjugation system-deficient cells. Those structures could fuse with lysosomes but the degradation of the inner autophagosomal membrane was significantly delayed. We suggest that the ATG conjugation-dependent closure of autophagosomes causes the inner autophagosomal membrane to become sensitive to lysosomal degradation.

The ATG conjugation systems are important for degradation of the inner autophagosomal membrane.

In macroautophagy, cytoplasmic contents are sequestered into the double-membrane autophagosome, which fuses with the lysosome to become the autolysosome. It has been thought that the autophagy-related (ATG) conjugation systems are required for autophagosome formation. Here, we found that autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 17-positive autophagosome-like structures could be generated even in the absence of the ATG conjugation systems, although at a reduced rate. These syntaxin 17-positive structures could further fuse with lysosomes, but degradation of the inner autophagosomal membrane was significantly delayed. Accordingly, autophagic activity in ATG conjugation-deficient cells was strongly suppressed. We suggest that the ATG conjugation systems, which are likely required for the closure (i.e., fission) of the autophagosomal edge, are not absolutely essential for autolysosome formation but are important for efficient degradation of the inner autophagosomal membrane.

Glutamatergic Monopolar Interneurons Provide a Novel Pathway of Excitation in the Mouse Retina.

Excitatory and inhibitory neurons in the CNS are distinguished by several features, including morphology, transmitter content, and synapse architecture [1]. Such distinctions are exemplified in the vertebrate retina. Retinal bipolar cells are polarized glutamatergic neurons receiving direct photoreceptor input, whereas amacrine cells are usually monopolar inhibitory interneurons with synapses almost exclusively in the inner retina [2]. Bipolar but not amacrine cell synapses have presynaptic ribbon-like structures at their transmitter release sites. We identified a monopolar interneuron in the mouse retina that resembles amacrine cells morphologically but is glutamatergic and, unexpectedly, makes ribbon synapses. These glutamatergic monopolar interneurons (GluMIs) do not receive direct photoreceptor input, and their light responses are strongly shaped by both ON and OFF pathway-derived inhibitory input. GluMIs contact and make almost as many synapses as type 2 OFF bipolar cells onto OFF-sustained A-type (AOFF-S) retinal ganglion cells (RGCs). However, GluMIs and type 2 OFF bipolar cells possess functionally distinct light-driven responses and may therefore mediate separate components of the excitatory synaptic input to AOFF-S RGCs. The identification of GluMIs thus unveils a novel cellular component of excitatory circuits in the vertebrate retina, underscoring the complexity in defining cell types even in this well-characterized region of the CNS.