RESUMO
Saccharomyces cerevisiae IA3 is a 68 amino acid peptide inhibitor of yeast proteinase A (YPRA) characterized as a random coil when in solution, folding into an N-terminal amphipathic alpha helix for residues 2-32 when bound to YPRA, with residues 33-68 unresolved in the crystal complex. Circular dichroism (CD) spectroscopy results show that amino acid substitutions that remove hydrogen-bonding interactions observed within the hydrophilic face of the N-terminal domain (NTD) of IA3-YPRA crystal complex reduce the 2,2,2-trifluoroethanol (TFE)-induced helical transition in solution. Although nearly all substitutions decreased TFE-induced helicity compared to wild-type (WT), each construct did retain helical character in the presence of 30% (v/v) TFE and retained disorder in the absence of TFE. The NTDs of 8 different Saccharomyces species have nearly identical amino acid sequences, indicating that the NTD of IA3 may be highly evolved to adopt a helical fold when bound to YPRA and in the presence of TFE but remain unstructured in solution. Only one natural amino acid substitution explored within the solvent-exposed face of the NTD of IA3 induced TFE-helicity greater than the WT sequence. However, chemical modification of a cysteine by a nitroxide spin label that contains an acetamide side chain did enhance TFE-induced helicity. This finding suggests that non-natural amino acids that can increase hydrogen bonding or alter hydration through side-chain interactions may be important to consider when rationally designing intrinsically disordered proteins (IDPs) with varied biotechnological applications.
Assuntos
Proteínas Intrinsicamente Desordenadas , Solventes , Proteínas Intrinsicamente Desordenadas/genética , Estrutura Secundária de Proteína , Ligação de Hidrogênio , Sequência de Aminoácidos , Saccharomyces cerevisiae , Dicroísmo Circular , Trifluoretanol/farmacologia , Dobramento de ProteínaRESUMO
The surface hydration diffusivity of Bacillus subtilis Lipase A (BSLA) has been characterized by low-field Overhauser dynamic nuclear polarization (ODNP) relaxometry using a series of spin-labeled constructs. Sites for spin-label incorporation were previously designed via an atomistic computational approach that screened for surface exposure, reflective of the surface hydration comparable to other proteins studied by this method, as well as minimal impact on protein function, dynamics, and structure of BSLA by excluding any surface site that participated in greater than 30% occupancy of a hydrogen bonding network within BSLA. Experimental ODNP relaxometry coupling factor results verify the overall surface hydration behavior for these BSLA spin-labeled sites similar to other globular proteins. Here, by plotting the ODNP parameters of relative diffusive water versus the relative bound water, we introduce an effective "phase-space" analysis, which provides a facile visual comparison of the ODNP parameters of various biomolecular systems studied to date. We find notable differences when comparing BSLA to other systems, as well as when comparing different clusters on the surface of BSLA. Specifically, we find a grouping of sites that correspond to the spin-label surface location within the two main hydrophobic core clusters of the branched aliphatic amino acids isoleucine, leucine, and valine cores observed in the BSLA crystal structure. The results imply that hydrophobic clustering may dictate local surface hydration properties, perhaps through modulation of protein conformations and samplings of the unfolded states, providing insights into how the dynamics of the hydration shell is coupled to protein motion and fluctuations.
Assuntos
Bacillus subtilis , Interações Hidrofóbicas e Hidrofílicas , Lipase , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ligação de Hidrogênio , Lipase/química , Lipase/metabolismo , Simulação de Dinâmica Molecular , Propriedades de Superfície , Água/químicaRESUMO
Spin-labeling with electron paramagnetic resonance spectroscopy (EPR) is a facile method for interrogating macromolecular flexibility, conformational changes, accessibility, and hydration. Within we present a computationally based approach for the rational selection of reporter sites in Bacillus subtilis lipase A (BSLA) for substitution to cysteine residues with subsequent modification with a spin-label that are expected to not significantly perturb the wild-type structure, dynamics, or enzymatic function. Experimental circular dichroism spectroscopy, Michaelis-Menten kinetic parameters and EPR spectroscopy data validate the success of this approach to computationally select reporter sites for future magnetic resonance investigations of hydration and hydration changes induced by polymer conjugation, tethering, immobilization, or amino acid substitution in BSLA. Analysis of molecular dynamic simulations of the impact of substitutions on the secondary structure agree well with experimental findings. We propose that this computationally guided approach for choosing spin-labeled EPR reporter sites, which evaluates relative surface accessibility coupled with hydrogen bonding occupancy of amino acids to the catalytic pocket via atomistic simulations, should be readily transferable to other macromolecular systems of interest including selecting sites for paramagnetic relaxation enhancement NMR studies, other spin-labeling EPR studies or any method requiring a tagging method where it is desirable to not alter enzyme stability or activity.
Assuntos
Bacillus subtilis , Lipase , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Marcadores de Spin , Espectroscopia de Ressonância MagnéticaRESUMO
IA3 is a 68 amino acid natural peptide/protein inhibitor of yeast aspartic proteinase A (YPRA) that is intrinsically disordered in solution with induced N-terminal helicity when in the protein complex with YPRA. Based on the intrinsically disordered protein (IDP) parameters of fractional net charge (FNC), net charge density per residue (NCPR), and charge patterning (κ), the two domains of IA3 are defined to occupy different domains within conformationally based subclasses of IDPs, thus making IA3 a bimodal domain IDP. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and low-field Overhauser dynamic nuclear polarization (ODNP) spectroscopy results show that these two domains possess different degrees of compaction and hydration diffusivity behavior. This work suggests that SDSL EPR line shapes, analyzed in terms of their local tumbling volume (VL), provide insights into the compaction of the unstructured IDP ensemble in solution and that protein sequence and net charge distribution patterns within a conformational subclass can impact bound water hydration dynamics, thus possibly offering an alternative thermodynamic property that can encode conformational binding and behavior of IDPs and liquid-liquid phase separations.
Assuntos
Proteínas Intrinsicamente Desordenadas , Saccharomyces cerevisiae , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Conformação Proteica , Marcadores de Spin , Sequência de Aminoácidos , Proteínas Intrinsicamente Desordenadas/químicaRESUMO
Sialoglycans on HeLa cells were labeled with a nitroxide spin radical through enzymatic glycoengineering (EGE)-mediated installation of azide-modified sialic acid (Neu5Ac9N3) and then click reaction-based attachment of a nitroxide spin radical. α2,6-Sialyltransferase (ST) Pd2,6ST and α2,3-ST CSTII were used for EGE to install α2,6- and α2,3-linked Neu5Ac9N3, respectively. The spin-labeled cells were analyzed by X-band continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to gain insights into the dynamics and organizations of cell surface α2,6- and α2,3-sialoglycans. Simulations of the EPR spectra revealed average fast- and intermediate-motion components for the spin radicals in both sialoglycans. However, α2,6- and α2,3-sialoglycans in HeLa cells possess different distributions of the two components, e.g., a higher average population of the intermediate-motion component for α2,6-sialoglycans (78%) than that for α2,3-sialoglycans (53%). Thus, the average mobility of spin radicals in α2,3-sialoglycans was higher than that in α2,6-sialoglycans. Given the fact that a spin-labeled sialic acid residue attached to the 6-O-position of galactose/N-acetyl-galactosamine would experience less steric hindrance and show more flexibility than that attached to the 3-O-position, these results may reflect the differences in local crowding/packing that restrict the spin-label and sialic acid motion for α2,6-linked sialoglycans. The studies further suggest that Pd2,6ST and CSTII may have different preferences for glycan substrates in the complex environment of the extracellular matrix. The discoveries of this work are biologically important as they are useful for interpreting the different functions of α2,6- and α2,3-sialoglycans and indicate the possibility of using Pd2,6ST and CSTII to target different glycoconjugates on cells.