DIGITAL NIST: An examination of the obstacles and opportunities in the digital transformation of NIST's reference materials.

Early in 2022, NIST embarked on a pilot project to produce digital calibration reports and digital certificates of analysis for reference materials. The goal is to produce examples of digital reports and certificates to assess the scope and challenges of digital transformation in those particular measurement services. This paper focuses on the Reference Material Certificate effort of the pilot project. Our aims for this part of the pilot project are: to generate a digital Reference Material Certificate from certification data; descriptive information about the material, and other data and metadata as needed; to generate a human-readable report from the digital Reference Material Certificate; and to hold a workshop to gather stakeholder feedback. The challenges for NIST include the diverse and complex information presently contained in NIST certificates, converting values to non-SI units to match the needs of stakeholders, and format updates to NIST Reference Material Certificates necessary to allow for machine generation. Other practical challenges include the wide variety of Reference Materials offered by NIST, as well as the needs of internal and external stakeholders. This presentation will report on the progress of the NIST effort and discuss some of the challenges and solutions to producing Digital Reference Material Certificates.

Polyelectrolyte domains and intrinsic disorder within the prismatic Asprich protein family.

A number of fully functional proteins have been identified to exist in a partially or fully disordered state. These intrinsically disordered proteins (IDP) are recognized as an important sequence class that fulfill many roles. A number of biomineral-associated proteins, particularly those which possess polyelectrolyte domains, represent potential members of the IDP class. This report describes a bioinformatics study of a ten member polyanionic sequence biomineralization protein family, Asprich, and the experimental characterization of the conserved N- and C-terminal regions found within seven members of this family. Using protein disorder prediction algorithms (DPROT, PONDR, GLOBPLOT), we confirm that all ten Asprich protein sequences are disordered, and that two polyelectrolyte domains within each protein contribute to the disorder scoring. Using synthetic peptides which model the conserved N- (F1,48 AA) and C-terminal (F2, 42 AA) domains, we determine that both domains are globally disordered and remain so in the presence of Ca(II). However, F1 and F2 possess differing proportions of extended beta strand relative to random coil structure and sequence spacing of Asp, Glu residues. As a result, the F2 sequence possesses a higher anionic surface charge density, solvent accessibility, and greater degree of local conformational response to Ca(II). These differences may explain why F1 and F2 differ with regard to step growth kinetics, mineral modulation, and metal ion complexation, and possibly distinguish the molecular role(s) that each domain conveys to the Asprich protein family. Structural and surface charge density features may also control the function of Asp, Glu polyelectrolyte domains within other IDP proteins.

The tooth enamel protein, porcine amelogenin, is an intrinsically disordered protein with an extended molecular configuration in the monomeric form.

Amelogenins make up a class of proteins associated with the formation of mineralized enamel in vertebrates, possess highly conserved N- and C-terminal sequence regions, and represent an interesting model protein system for understanding biomineralization and protein assembly. Using bioinformatics, we report here the identification of molecular traits that classify 12 amelogenin proteins as members of the intrinsically disordered or unstructured protein family (IDPs), a group of proteins that normally exist as unfolded species but are capable of transformation to a folded state as part of their overall function. Using biophysical techniques (CD and NMR), we follow up on our bioinformatics studies and confirm that one of the amelogenins, recombinant porcine rP172, exists in an extended, unfolded state in the monomeric form. This protein exhibits evidence of conformational exchange between two states, and this exchange may be mediated by Pro residues in the sequence. Although the protein is globally unfolded, we detect the presence of local residual secondary structure [alpha-helix, extended beta-strand, turn/loop, and polyproline type II (PPII)] that may serve several functional roles within the enamel matrix. The extended, labile conformation of rP172 amelogenin is compatible with the known functions of amelogenin in enamel biomineralization, i.e., self-assembly, associations with other enamel matrix proteins and with calcium phosphate biominerals, and interaction with cell receptors. It is likely that the labile structure of this protein facilitates interactions of amelogenin with other macromolecules or with minerals for achievement of internal protein stabilization.

Probing the organic-mineral interface at the molecular level in model biominerals.

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.

Expected and unexpected effects of amino acid substitutions on polypeptide-directed crystal growth.

Nature's use of biomineralization polypeptides to control and modulate the growth of biogenic minerals is an important process that, if properly understood, could have significant implications for designing and creating new inorganic-based materials. Although the sequences for a number of biomineralization proteins exist, very little is known about the participation of specific amino acids in the mineral modulation process. In this letter, we investigate the impact of global Asp --> Asn and Glu --> Gln substitutions on two mollusk shell nacre polypeptides, AP7N and n16N. We find that these global substitutions, which remove all anionic Ca(II) binding sites, abolish the expected in vitro mineralization activities associated with each native polypeptide. In addition, the ability of substituted peptides to form complexes with both Ca(II) and Ca(II) metal ion analogs is also abolished. However, some unexpected effects were noted. First, the Asp --> Asn, Glu --> Gln substituted n16N polypeptide is observed to self-assemble and form biofilms or coatings that appear to mineralize in vitro. Second, both polypeptides are structurally affected by these substitutions, with Asp --> Asn substituted AP7N transforming to an alpha helix and Asp --> Asn, Glu --> Gln substituted n16N transforming to a more unfolded random-coil-like structure. We find that the participation of Asp and Glu residues is crucial to the inherent mineralization activities and conformations of AP7N and n16N polypeptides. Surprisingly, we find that the replacement of anionic residues within biomineralization polypeptides such as n16N still permits mineral modulation, but in a different form that now involves peptide self-association and biofilm formation.

Mechanisms of amine-catalyzed organosilicate hydrolysis at circum-neutral pH.

Mono- and polyamines can catalyze the hydrolysis and condensation of organosilicate starting materials in biomimetic silica synthesis pathways at circum-neutral pHs and room temperature. Our study is focused on understanding the mechanistic role of amines in catalyzing the hydrolysis process that precedes condensation. We have conducted (29)Si NMR experimental studies over a range of temperature and pHs for the hydrolysis rates of trimethylethoxysilane (TMES), a model compound with only one hydrolyzable bond, combined with quantum mechanical hybrid density functional theory calculations of putative intermediate and transition-state structures for TMES and tetramethyl orthosilicate (TMOS). Comparison of calculated energies with experimentally determined activation energies indicates that amine catalysis of TMES is primarily a consequence of the amine's acidity at neutral pH. The proton released by the amine is transferred to the organosilicate, producing a protonated ethoxy leaving group that can be displaced by water in an S(N)2 reaction. For TMOS, the activation energy of proton-transfer coupled with S(N)2 substitution is comparable to that for Corriu's nucleophile-activated nucleophilic displacement, such that the mechanism of amine-catalyzed hydrolysis is dependent mostly on the ambient pH conditions as well as the type of amine. The relevance of our results to biological silica precipitation is discussed.