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 NMRbased 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

A NMR protocol is introduced that permits accurate measurement of minute, remote chemical shift perturbations (CSPs), caused by a mutation-induced change in the electric field. Using protein GB3 as a model system, 1HN CSPs in K19A and K19E mutants can be fitted to small changes in the electric field at distal sites in the protein using the Buckingham equation, yielding an apparent dielectric constant εa of 8.6 ± 0.8 at 298 K. These CSPs, and their derived εa value, scale strongly with temperature. For example, CSPs at 313 K are about ∼30% smaller than those at 278 K, corresponding to an effective εa value of about 7.3 at 278 K and 10.5 at 313 K. Molecular dynamics simulations in explicit solvent indicate that solvent water makes a significant contribution to εa.

The intramolecular electric field (e-field) generated by protein GB3 side-chain charges K/E10, K/E19, and D/K40 was measured in the absence or presence of macromolecular crowding. The e-field responds differently to different crowding agents-dextran, Ficoll, BSA, and E. coli cell lysate. Dextran and Ficoll have no effect on the e-field. The lysate generally weakens the e-field but the amplitude of weakening varies greatly. For example, the e-field by K19 is reduced by 67% in the presence of 90 g/L lysate, corresponding to a charge change from 0.9 to 0.3 e for K19, whereas the e-fields by D/K40 are weakened only by ∼7% under the same lysate concentration. The extent of the e-field weakening by BSA is in between that by Ficoll (dextran) and lysate. Further investigations suggest that the e-field weakening mechanism by lysate is similar to that by NaCl. That is, the e-field generated by a protein surface charge affects the distribution of lysate which creates a reaction field and weakens the protein e-field. Our study indicates that the protein electrostatic property can be changed significantly due to quinary interaction with the cell environment.

Biofilms are accumulations of microorganisms embedded in extracellular matrices that protect against external factors and stressful environments. Cyanobacterial biofilms are ubiquitous and have potential for treatment of wastewater and sustainable production of biofuels. But the underlying mechanisms regulating cyanobacterial biofilm formation are unclear. Here, we report the solution NMR structure of a protein, Se0862, conserved across diverse cyanobacterial species and involved in regulation of biofilm formation in the cyanobacterium Synechococcus elongatus PCC 7942. Se0862 is a class α+β protein with ααββββαα topology and roll architecture, consisting of a fourstranded β-sheet that is flanked by four α-helices on one side. Conserved surface residues constitute a hydrophobic pocket and charged regions that are likely also present in Se0862 orthologs.