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Ancestral reconstruction provides instrumental insights regarding the biochemical and biophysical characteristics of past proteins. A striking observation relates to the remarkably high thermostability of reconstructed ancestors. The latter has been linked to high environmental temperatures in the Precambrian era, the era relating to most reconstructed proteins. We found that inferred ancestors of the serum paraoxonase (PON) enzyme family, including the mammalian ancestor, exhibit dramatically increased thermostabilities compared with the extant, human enzyme (up to 30 °C higher melting temperature). However, the environmental temperature at the time of emergence of mammals is presumed to be similar to the present one. Additionally, the mammalian PON ancestor has superior folding properties (kinetic stability)—unlike the extant mammalian PONs, it expresses in E. coli in a soluble and functional form, and at a high yield. We discuss two potential origins of this unexpectedly high stability. First, ancestral stability may be overestimated by a “consensus effect,” whereby replacing amino acids that are rare in contemporary sequences with the amino acid most common in the family increases protein stability. Comparison to other reconstructed ancestors indicates that the consensus effect may bias some but not all reconstructions. Second, we note that high stability may relate to factors other than high environmental temperature such as oxidative stress or high radiation levels. Foremost, intrinsic factors such as high rates of genetic mutations and/or of transcriptional and translational errors, and less efficient protein quality control systems, may underlie the high kinetic and thermodynamic stability of past proteins.

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO₂. Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO₂. Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO₂ release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic–stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO₂, even if most of their enzymes operate at a tenth of Rubisco’s maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD⁺, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway.

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90 degree C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.

When holding a tablet computer with two hands, the touch keyboard configuration imposes postural constraints on the user because of the need to simultaneously hold the device and type with the thumbs. Designers have provided users with several possible keyboard configurations (device orientation, keyboard layout and location). However, potential differences in performance, usability and postures among these configurations have not been explored. We hypothesize that (1) the narrower standard keyboard layout in the portrait orientation leads to lower self-reported discomfort and less reach than the landscape orientation; (2) a split keyboard layout results in better overall outcomes compared to the standard layout; and (3) the conventional bottom keyboard location leads to the best outcomes overall compared to other locations. A repeated measures laboratory experiment of 12 tablet owners measured typing speed, discomfort, task difficulty, and thumb/wrist joint postures using an active marker system during typing tasks for different combinations of device orientation (portrait and landscape), keyboard layout (standard and split), and keyboard location (bottom, middle, top). The narrower standard keyboard with the device in the portrait orientation was associated with less discomfort (least squares mean (and S.E.) 2.9±0.6) than the landscape orientation (4.5±0.7). Additionally, the split keyboard decreased the amount of reaching required by the thumb in the landscape orientation as defined by a reduced range of motion and less MCP extension, which may have led to reduced discomfort (2.7±0.6) compared to the standard layout (4.5±0.7). However, typing speed was greater for the standard layout (127±5 char./min.) compared to the split layout (113±4 char./min.) regardless of device orientation and keyboard location. Usage guidelines and designers can incorporate these findings to optimize keyboard design parameters and form factors that promote user performance and usability for thumb interaction.

Design features of mobile computing technology such as device size and key location may affect thumb motor performance during single-handed use. Since single-handed use requires the thumb posture to vary with key location, we hypothesize that motor performance is associated with thumb and wrist joint postures. A repeated measures laboratory experiment of 10 right-handed participants measured thumb and wrist joint postures during reciprocal tapping tasks between two keys for different key pairs among 12 emulated keys. Fitts' effective index of performance and joint postures at contact with each key were averaged across trials for each key. Thumb motor performance varied for different keys, with poorest performances being associated with excessive thumb flexion such as when tapping on keys closest to the base of the thumb in the bottom right corner of the phone. Motor performance was greatest when the thumb was in a typical resting posture, neither significantly flexed nor fully extended with slight CMC joint abduction and supination, such as when tapping on keys located in the top right and middle left areas on the phone. Grip was also significantly affected by key location, with the most extreme differences being between the top left and bottom right corners of the phone. These results suggest that keypad designs aimed at promoting performance for single-handed use should avoid placing frequently used functions and keys close to the base of the thumb and instead should consider key locations that require a thumb posture away from its limits in flexion/extension, as these postures promote motor performance.

Thumb interaction is a primary technique used to operate small handheld devices such as smartphones. Despite the different techniques involved in operating a handheld device compared to a personal computer, the keyboard layouts for both devices are similar. A handheld device keyboard that considers the physical capabilities of the thumb may improve user experience. We developed and applied a design evaluation tool for different geometries of the QWERTY keyboard using a performance evaluation model. The model utilizes previously collected data on thumb motor performance and posture for different tap locations and thumb movement directions. We calculated a performance index (PITOT, 0 is worst and 2 is best) for 663 designs consisting in different combinations of three variables: the keyboard's radius of curvature (R) (mm), orientation (O) (°), and vertical location on the screen (L). The current standard keyboard performed poorly (PITOT = 0.28) compared to other designs considered. Keyboard location (L) contributed to the greatest variability in performance out of the three design variables, suggesting that designers should modify this variable first. Performance was greatest for designs in the middle keyboard location. In addition, having a slightly upward curve (R = -20 mm) and orientated perpendicular to the thumb's long axis (O = -20°) improved performance to PITOT = 1.97. Poorest performances were associated with placement of the keyboard's spacebar in the bottom right corner of the screen (e.g., the worst was for R = 20 mm, O = 40°, L =  Bottom (PITOT = 0.09)). While this evaluation tool can be used in the design process as an ergonomic reference to promote user motor performance, other design variables such as visual access and usability still remain unexplored.

Thumb interaction is a primary technique used to operate small handheld devices such as smartphones. Despite the different techniques involved in operating a handheld device compared to a personal computer, the keyboard layouts for both devices are similar. A handheld device keyboard that considers the physical capabilities of the thumb may improve user experience. We developed and applied a design evaluation tool for different geometries of the QWERTY keyboard using a performance evaluation model. The model utilizes previously collected data on thumb motor performance and posture for different tap locations and thumb movement directions. We calculated a performance index (PITOT, 0 is worst and 2 is best) for 663 designs consisting in different combinations of three variables: the keyboard's radius of curvature (R) (mm), orientation (O) ( degree ), and vertical location on the screen (L). The current standard keyboard performed poorly (PITOT = 0.28) compared to other designs considered. Keyboard location (L) contributed to the greatest variability in performance out of the three design variables, suggesting that designers should modify this variable first. Performance was greatest for designs in the middle keyboard location. In addition, having a slightly upward curve (R = -20 mm) and orientated perpendicular to the thumb's long axis (O = -20 degree ) improved performance to PITOT = 1.97. Poorest performances were associated with placement of the keyboard's spacebar in the bottom right corner of the screen (e.g., the worst was for R = 20 mm, O = 40 degree , L = Bottom (PITOT = 0.09)). While this evaluation tool can be used in the design process as an ergonomic reference to promote user motor performance, other design variables such as visual access and usability still remain unexplored.