California's experience with SMA newborn screening: A successful path to early intervention.

BACKGROUND: Universal spinal muscular atrophy (SMA) newborn screening was implemented in California on June 24, 2020. OBJECTIVE: We describe California's experience with the first 18 months of SMA newborn screening, including our assay methodology, timeliness of screening and follow-up milestones, and clinical and epidemiological outcomes observed. METHODS: Dried blood spots are screened for SMA using multiplex real time polymerase chain reaction (RT-PCR) to detect deletions of exon 7 in the survival of motor neuron 1 (SMN1) gene. Short-term follow-up data is collected from clinical staff via an online data collection tool. RESULTS: In the first 18 months, 628,791 newborns from California's diverse population were tested for SMA. Thirty-four screened positive and were confirmed to have the disorder. Infants were referred, diagnosed, and treated at a median of 8, 12, and 33 days of life, respectively. Nearly all infants received the desired treatment modality, and 62% received treatment while still asymptomatic. CONCLUSIONS: SMA newborn screening is a highly sensitive and specific test which identifies infants with SMA early when treatment is most effective. Even with newborn screening's success in facilitating early intervention, there is still work to be done to expedite treatment, especially for infants with the most severe form of the disease.

The Importance of the Scaffold for de Novo Enzymes: A Case Study with Kemp Eliminase.

We report electric field values relevant to the reactant and transition states of designed Kemp eliminases KE07 and KE70 and their improved variants from laboratory directed evolution (LDE), using atomistic simulations with the AMOEBA polarizable force field. We find that the catalytic base residue contributes the most to the electric field stabilization of the transition state of the LDE variants of the KE07 and KE70 enzymes, whereas the electric fields of the remainder of the enzyme and solvent disfavor the catalytic reaction in both cases. By contrast, we show that the electrostatic environment plays a large and stabilizing role for the naturally occurring enzyme ketosteroid isomerase (KSI). These results suggest that LDE is ultimately a limited strategy for improving de novo enzymes since it is largely restricted to optimization of chemical positioning in the active site, thus yielding a ∼3 order magnitude improvement over the uncatalyzed reaction, which we suggest may be an absolute upper bound estimate based on LDE applied to comparable de novo Kemp eliminases and other enzymes like KSI. Instead de novo enzymatic reactions could more productively benefit from optimization of the electrostatics of the protein scaffold in early stages of the computational design, utilizing electric field optimization as guidance.

The role of side chain entropy and mutual information for improving the de novo design of Kemp eliminases KE07 and KE70.

Side chain entropy and mutual entropy information between residue pairs have been calculated for two de novo designed Kemp eliminase enzymes, KE07 and KE70, and for their most improved versions at the end of laboratory directed evolution (LDE). We find that entropy, not just enthalpy, helped to destabilize the preference for the reactant state complex of the designed enzyme as well as favoring stabilization of the transition state complex for the best LDE enzymes. Furthermore, residues with the highest side chain couplings as measured by mutual information, when experimentally mutated, were found to diminish or annihilate catalytic activity, some of which were far from the active site. In summary, our findings demonstrate how side chain fluctuations and their coupling can be an important design feature for de novo enzymes, and furthermore could be utilized in the computational steps in lieu of or in addition to the LDE steps in future enzyme design projects.

A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ß-amyloid peptides from E. coli for NMR-based structural analysis.

Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized ß-amyloid peptides with Aß42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aß42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aß42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aß42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aß42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aß42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aß42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aß42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aß42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aß42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aß42 peptide as well as its other variants including any Aß42 peptide mutants.

Kinetic Detection of Orthogonal Protein and Chemical Coordinates in Enzyme Catalysis: Double Mutants of Soybean Lipoxygenase.

Soybean lipoxygenase-1 (SLO-1) is a paradigmatic enzyme system for studying the contribution of hydrogen tunneling to enzymatic proton-coupled electron transfer processes. In this study, the impact of pairs of double mutants on the properties of SLO-1 is presented. Steady-state rates and their deuterium kinetic isotope effects (KIEs) have been measured for the bimolecular reaction of enzyme with free substrate (kcat/Km) and compared to the unimolecular rate constant, kcat. A key kinetic finding is that the competitive KIEs on the second-order rate constant (kcat/Km) are all reduced from (D)kcat and, despite large changes in rate and activation parameters, remain essentially unaltered under a variety of conditions. These data implicate a protein reaction coordinate that is orthogonal to the chemical reaction coordinate and controls the concentration of the active enzyme. This study introduces a new means to interrogate the alteration of conformational landscapes that can occur following site-specific mutagenesis.

Extremely elevated room-temperature kinetic isotope effects quantify the critical role of barrier width in enzymatic C-H activation.

The enzyme soybean lipoxygenase (SLO) has served as a prototype for hydrogen-tunneling reactions, as a result of its unusual kinetic isotope effects (KIEs) and their temperature dependencies. Using a synergy of kinetic, structural, and theoretical studies, we show how the interplay between donor-acceptor distance and active-site flexibility leads to catalytic behavior previously predicted by quantum tunneling theory. Modification of the size of two hydrophobic residues by site-specific mutagenesis in SLO reduces the reaction rate 10(4)-fold and is accompanied by an enormous and unprecedented room-temperature KIE. Fitting of the kinetic data to a non-adiabatic model implicates an expansion of the active site that cannot be compensated by donor-acceptor distance sampling. A 1.7 Å resolution X-ray structure of the double mutant further indicates an unaltered backbone conformation, almost identical side-chain conformations, and a significantly enlarged active-site cavity. These findings show the compelling property of room-temperature hydrogen tunneling within a biological context and demonstrate the very high sensitivity of such tunneling to barrier width.

T7 phage display as a method of peptide ligand discovery for PDZ domain proteins.

The use of bacteriophage T7 is presented as a peptide display platform to identify short binding sequences for PDZ domain proteins. Two different domains are examined, the 10th PDZ domain (PDZ10) of the multi-PDZ domain protein 1 (MUPP1) and the third PDZ domain (PDZ3) of postsynaptic density-95 (PSD-95) protein. Using the T7Select 415-1b construct, which displays 415 peptides per phage particle, a random heptapeptide and focused octapeptide libraries were constructed and subjected to iterative selection-enrichment cycles against surface-immobilized PDZ3 and PDZ10 proteins. The derived consensus sequences, together with those of high-frequency clones, were used as the basis for individual chemically synthesized peptides. Each peptide was subjected to isothermal titration calorimetry binding determinations against the corresponding PDZ domain under standard solution conditions. For MUPP1 PDZ10, binding analysis demonstrated that one of the heptapeptides, Ac-IGRISRV, displayed a two-fold improved affinity over the octapeptide derived from the carboxy terminus of the hc-Kit protein, which we had recently demonstrated as among the highest affinity ligands reported to date for that domain. In the case of PSD-95 PDZ3, peptides were found that possessed low-micromolar dissociation constants, as well as those that rediscovered the C-terminal sequence (KQTSV) of the protein CRIPT, a known natural binding protein of PDZ3. These successful examples of ligand discovery against two distinctly different PDZ domains demonstrate that the T7 phage platform could prove broadly applicable to the numerous other PDZ domains for which binding peptides are absent or of insufficient affinity.

Experimental evidence for hydrogen tunneling when the isotopic arrhenius prefactor (A(H)/A(D)) is unity.

The temperature dependence of the kinetic isotope effect (KIE) is one of the major tools used for the investigation of hydrogen tunneling in condensed phase. Hydrogen transfer reactions displaying isotopic Arrhenius prefactor ratios (A(H)/A(D)) of unity are generally ascribed to a semiclassical mechanism. Here, we have identified a double mutant of soybean lipoxygenase (SLO-1, an enzyme previously shown to follow quantum mechanical hydrogen tunneling), that displays an A(H)/A(D) of unity and highly elevated (nonclassical) KIEs. This observation highlights the shortcoming of assigning a hydrogen transfer reaction to a semiclassical model based solely on an Arrhenius prefactor ratio.

Design, synthesis, and evaluation of linear and cyclic peptide ligands for PDZ10 of the multi-PDZ domain protein MUPP1.

PDZ10 is the 10th of 13 PDZ domains found within MUPP1, a cytoplasmic scaffolding protein first identified as an endogenous binding partner of serotonin receptor type 2c (5HT2c). This association, as with those of several other interacting proteins that have subsequently been identified, is mediated through the C-terminal tail of the PDZ domain partner. Using isothermal titration calorimetry (ITC), we measured the thermodynamic binding parameters [changes in Gibbs free energy (DeltaG), enthalpy (DeltaH) and entropy (TDeltaS)] of the isolated PDZ10 domain for variable-length N-acetylated peptides from the 5HT2c serotonin receptor C-terminal sequence, as well as for octapeptides of eight other putative partner proteins of PDZ10 (5HT2a, hc-kit, hTapp1, mTapp2, TARP, NG2, claudin-1, and HPV-18 E6). In length dependence studies of the 5HT2c sequence, the maximal affinity of the peptides leveled off rapidly and further elongation did not significantly improve the dissociation constant (Kd) of 11 microM observed with the pentapeptide. Among the native partners of PDZ10, octapeptides derived from the hc-kit and 5HT2c proteins were the strongest binders, with Kd values of 5.2 and 8.5 microM, respectively. The heat capacity change (DeltaCp) for the 5HT2c octapeptide was determined to be -94 cal/mol, and a calculated estimate indicates burial of polar and apolar surface areas in equal measure upon ligand binding. Peptides with phosphoserine at either the P-1 or P-2 position experienced decreased affinity, which is in accord with the hypothesis that reversible phosphorylation is a possible mechanism for regulating PDZ domain-mediated interactions. Additionally, two conformationally constrained side chain-bridged cyclic peptide ligands were also designed, prepared, evaluated by ITC, and shown to bind PDZ10 primarily through a favorable change in entropy.