NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR‐derived contacts for an integral membrane protein (T1088). For the three targets with NMR‐based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of “inverse structure determination,” in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238‐residue integral membrane porin, was also used to assess several NMR‐assisted prediction methods. Most groups involved in this exercise generated similar beta‐barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Metamorphic proteins such as circadian clock protein KaiB and human chemokine XCL1 play vital roles in regulating biological processes, including gene expression, circadian clock and innate immune responses, and perform distinct functions in living cell by switching different structures in response to cellular environment stimuli. However, it is unclear how complex and crowded intracellular environments affect conformational rearrangement of metamorphic proteins. Here, the kinetics and thermodynamics of two well-characterized metamorphic proteins, circadian clock protein KaiB and human chemokine XCL1, were quantified in physiologically relevant environments by using NMR spectroscopy, indicating that crowded agents shift equilibrium towards the inactive form (ground-state KaiB and Ltn10-like state XCL1) without disturbing the corresponding structures, and crowded agents have predominantly impact on the exchange rate of XCL1 that switches folds on timescales of seconds, but have slightly impact on the exchange rate of KaiB that switches folds on timescales of hours. Our data shed light on how metamorphic proteins can respond immediately to the changed crowded intracellular conditions that induced by environmental cues and then execute different functions in living cell, and it also enhances our understanding of how environments enrich the sequence-structure-function paradigm.

The oscillator of the cyanobacterial circadian clock relies on the ability of the KaiB protein to switch reversibly between a stable ground-state fold (gsKaiB) and an unstable fold-switched fold (fsKaiB). Rare fold-switching events by KaiB provide a critical delay in the negative feedback loop of this posttranslational oscillator. In this study, we experimentally and computationally investigate the temperature dependence of fold switching and its mechanism. We demonstrate that the stability of gsKaiB increases with temperature compared to fsKaiB and that the Q10 value for the gsKaiB → fsKaiB transition is nearly three times smaller than that for the reverse transition in a construct optimized for NMR studies. Simulations and native-state hydrogen-deuterium exchange NMR experiments suggest that fold switching can involve both partially and completely unfolded intermediates. The simulations predict that the transition state for fold switching coincides with isomerization of conserved prolines in the most rapidly exchanging region, and we confirm experimentally that proline isomerization is a rate-limiting step for fold switching. We explore the implications of our results for temperature compensation, a hallmark of circadian clocks, through a kinetic model.

The protein Griffithsin (Grft) is a lectin that tightly binds to high-mannose glycosylation sites on viral surfaces. This property allows Grft to potently inhibit many viruses, including HIV-1. The major route of HIV infection is through sexual activity, so an important tool for reducing the risk of infection would be a film that could be inserted vaginally or rectally to inhibit transmission of the virus. We have previously shown that silk fibroin can encapsulate, stabilize, and release various antiviral proteins, including Grft. However, for broad utility as a prevention method, it would be useful for an insertable film to adhere to the mucosal surface so that it remains for several days or weeks to provide longer-term protection from infection. We show here that silk fibroin can be formulated with adhesive properties using the nontoxic polymer hydroxypropyl methylcellulose (HPMC) and glycerol, and that the resulting silk scaffold can both adhere to biological surfaces and release Grft over the course of at least one week. This work advances the possible use of silk fibroin as an anti-viral insertable device to prevent infection by sexually transmitted viruses, including HIV-1.

In order to improve machine performance, the optics parameters of the storage ring need to be measured accurately. The commonly used accelerator optics parameters, including momentum compaction factor and dispersion function, are key parameters that reflect the state of the machine. It is difficult to measure them with traditional methods during the user operation run. Based on a high-resolution bunch-by-bunch three-dimensional measurement system, a noninvasive measurement of momentum compaction factor and the dispersion function was performed at the Shanghai synchrotron radiation facility (SSRF). The dispersion function can be obtained purely from the direct observation results of the bunch-by bunch 3D position, based on the definition of the dispersion function during normal user operation run without perturbing the beam. The theoretical background and experimental results of the method are discussed in this paper. The measurement results are in good agreement with the design values.