scholarly journals Identification of biochemically neutral positions in liver pyruvate kinase

2019 ◽  
Author(s):  
Tyler A. Martin ◽  
Tiffany Wu ◽  
Qingling Tang ◽  
Larissa L. Dougherty ◽  
Daniel J. Parente ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Protein Function ◽  
Functional Outcomes ◽  
High Surface ◽  
Experimental Studies ◽  
Substitution Effects ◽  
Evolutionary Patterns ◽  
Allosteric Coupling ◽  
Allosteric Binding Sites ◽  
Residue Position

AbstractUnderstanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs. does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions – high sequence entropy, high surface exposure, and alanine scanning – lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a “neutrality” score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

scholarly journals Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tiffany Wu ◽  
Liskin Swint-Kruse ◽  
Aron W. Fenton
Keyword(s):  
Binding Sites ◽  
Functional Outcomes ◽  
Human Liver ◽  
Full Range ◽  
Allosteric Coupling ◽  
Multiple Parameters ◽  
Wide Range ◽  
Allosteric Binding Sites ◽  
Fructose 1,6 Bisphosphate ◽  
Effector Binding

AbstractFor protein mutagenesis, a common expectation is that important positions will behave like on/off “toggle” switches (i.e., a few substitutions act like wildtype, most abolish function). However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: “rheostat” positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostatic effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of “local” positions, i.e., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allosterically-coupled binding sites. Positions near the Ala binding site had rheostatic outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.

scholarly journals Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites

2019 ◽  
Author(s):  
Tiffany Wu ◽  
Liskin Swint-Kruse ◽  
Aron W. Fenton
Keyword(s):  
Binding Sites ◽  
Functional Outcomes ◽  
Human Liver ◽  
Full Range ◽  
Allosteric Coupling ◽  
Multiple Parameters ◽  
Wide Range ◽  
Allosteric Binding Sites ◽  
Fructose 1,6 Bisphosphate ◽  
Effector Binding

AbstractFor protein mutagenesis, a common expectation is that important positions will behave like on/off “toggle” switches (i.e., a few substitutions act like wildtype, most abolish function). However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: “rheostat” positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostat effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of “local” positions, i.e., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allosterically-coupled binding sites. Positions near the Ala binding site had rheostat outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.

scholarly journals Chemical Decorations of “MARs” Residents in Orchestrating Eukaryotic Gene Regulation

2020 ◽  
Vol 8 ◽  
Author(s):  
Tanaya Roychowdhury ◽  
Samit Chattopadhyay
Keyword(s):  
Gene Regulation ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Protein Function ◽  
Functional Outcomes ◽  
Three Dimensional ◽  
Limited Information ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
3D Matrix

Genome organization plays a crucial role in gene regulation, orchestrating multiple cellular functions. A meshwork of proteins constituting a three-dimensional (3D) matrix helps in maintaining the genomic architecture. Sequences of DNA that are involved in tethering the chromatin to the matrix are called scaffold/matrix attachment regions (S/MARs), and the proteins that bind to these sequences and mediate tethering are termed S/MAR-binding proteins (S/MARBPs). The regulation of S/MARBPs is important for cellular functions and is altered under different conditions. Limited information is available presently to understand the structure–function relationship conclusively. Although all S/MARBPs bind to DNA, their context- and tissue-specific regulatory roles cannot be justified solely based on the available information on their structures. Conformational changes in a protein lead to changes in protein–protein interactions (PPIs) that essentially would regulate functional outcomes. A well-studied form of protein regulation is post-translational modification (PTM). It involves disulfide bond formation, cleavage of precursor proteins, and addition or removal of low-molecular-weight groups, leading to modifications like phosphorylation, methylation, SUMOylation, acetylation, PARylation, and ubiquitination. These chemical modifications lead to varied functional outcomes by mechanisms like modifying DNA–protein interactions and PPIs, altering protein function, stability, and crosstalk with other PTMs regulating subcellular localizations. S/MARBPs are reported to be regulated by PTMs, thereby contributing to gene regulation. In this review, we discuss the current understanding, scope, disease implications, and future perspectives of the diverse PTMs regulating functions of S/MARBPs.

scholarly journals A Mucoadhesive Electrospun Nanofibrous Matrix for Rapid Oramucosal Drug Delivery

2013 ◽  
Vol 2013 ◽  
pp. 1-19 ◽  
Author(s):  
Clare Dott ◽  
Charu Tyagi ◽  
Lomas K. Tomar ◽  
Yahya E. Choonara ◽  
Pradeep Kumar ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Adhesion Force ◽  
High Surface ◽  
Experimental Studies ◽  
Volume Ratio ◽  
Work Of Adhesion ◽  
Computer Assisted ◽  
Glycerol Concentration ◽  
Disintegration Time ◽  
Fill Volume

A nanofibrous matrix system (NFMS), consisting of a drug-loaded nanofiber layer, was electrospun directly onto a polymeric backing film, the latter of which was formulated and optimized according to a 3-level, 3-factor Box-Behnken experimental design. The dependent variables, fill volume, hydroxypropylmethylcellulose (HPMC) concentration, and glycerol concentration, were assessed for their effects on measured responses, disintegration time, work of adhesion, force of adhesion, dissolution area under curve (AUC) at 1 minute, and permeation AUC at 3 minutes. Physicochemical and physicomechanical properties of the developed system were studied by rheology, FTIR, toughness determination, mucoadhesion, and nanotensile testing. Data obtained from the physicomechanical characterization confirmed the suitability of NFMS for application in oramucosal drug delivery. The optimized NFMS showed the drug entrapment of 2.3 mg/1.5 cm2with disintegration time of 12.8 seconds. Electrospinning of drug-loaded polyvinylalcohol (PVA) fibers resulted in a matrix with an exceedingly high surface-area-to-volume ratio, which enhanced the rate of dissolution for rapid oramucosal drug delivery. To corroborate with the experimental studies, the incorporation of glycerol with HPMC and PVA blend was mechanistically elucidated using computer-assisted modeling of the 3D polymeric architecture of the respective molecular complexes to envisage the likely alignment of the polymer morphologies affecting the performance of the nanofibrous device.

Filler Aggregates and Their Effect on Reinforcement

1974 ◽  
Vol 47 (2) ◽  
pp. 411-433 ◽  
Author(s):  
A. I. Medalia
Keyword(s):  
Carbon Black ◽  
Fine Particles ◽  
High Surface ◽  
High Surface Area ◽  
Experimental Studies ◽  
Spherical Particles ◽  
Rubber Compound ◽  
Carbon Blacks ◽  
Reinforcing Fillers ◽  
Aggregate Nature

Abstract The most highly reinforcing fillers, namely carbon blacks and silicas, consist of aggregates of quasi-spherical particles fused together. In the absence of direct experimental studies with single-particle carbon blacks or silicas of high surface area, we cannot be sure if aggregated structure is essential for good reinforcement, or whether aggregation and fusion just happen to accompany the formation of fine particles at practical concentrations. In any case, there is no doubt that the aggregate nature of the filler plays a major role in determining the properties of the rubber compound. Here I would like to review what we know about filler aggregates, especially of carbon black, and suggest some mechanisms for their effects on rubber; and also indicate where our knowledge seems inadequate at the present time.

scholarly journals Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins

2021 ◽  
Vol 22 (14) ◽  
pp. 7267
Author(s):  
Léni Jodaitis ◽  
Thomas van Oene ◽  
Chloé Martens
Keyword(s):  
Membrane Proteins ◽  
Molecular Mechanism ◽  
Protein Interactions ◽  
Protein Function ◽  
Biological Membrane ◽  
Allosteric Coupling ◽  
Lipid Protein Interactions ◽  
Experimental Challenge ◽  
Membrane Protein Function

Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid–protein interactions in the mechanism of membrane proteins.

scholarly journals Stearic acid blunts growth-factor signaling via oleoylation of GNAI proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hana Nůsková ◽  
Marina V. Serebryakova ◽  
Anna Ferrer-Caelles ◽  
Timo Sachsenheimer ◽  
Christian Lüchtenborg ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Protein Function ◽  
Functional Outcomes ◽  
Covalent Attachment ◽  
Growth Factor Signaling ◽  
Egfr Signaling ◽  
Akt Activation ◽  
Functional Consequences ◽  
Protein Acylation

AbstractCovalent attachment of C16:0 to proteins (palmitoylation) regulates protein function. Proteins are also S-acylated by other fatty acids including C18:0. Whether protein acylation with different fatty acids has different functional outcomes is not well studied. We show here that C18:0 (stearate) and C18:1 (oleate) compete with C16:0 to S-acylate Cys3 of GNAI proteins. C18:0 becomes desaturated so that C18:0 and C18:1 both cause S-oleoylation of GNAI. Exposure of cells to C16:0 or C18:0 shifts GNAI acylation towards palmitoylation or oleoylation, respectively. Oleoylation causes GNAI proteins to shift out of cell membrane detergent-resistant fractions where they potentiate EGFR signaling. Consequently, exposure of cells to C18:0 reduces recruitment of Gab1 to EGFR and reduces AKT activation. This provides a molecular mechanism for the anti-tumor effects of C18:0, uncovers a mechanistic link how metabolites affect cell signaling, and provides evidence that the identity of the fatty acid acylating a protein can have functional consequences.

scholarly journals Aromatic interactions with membrane modulate human BK channel activation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Mahdieh Yazdani ◽  
Guohui Zhang ◽  
Zhiguang Jia ◽  
Jingyi Shi ◽  
Jianmin Cui ◽  
...  
Keyword(s):  
Protein Function ◽  
Bk Channels ◽  
Bk Channel ◽  
Atomistic Simulations ◽  
Allosteric Coupling ◽  
Linker Sequence ◽  
Aromatic Interactions ◽  
Sequence Scrambling ◽  
Nonspecific Interactions ◽  
Potential Complex

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue ‘C-linker’. To determine the linker’s role in human BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

scholarly journals Nonspecific membrane interactions can modulate BK channel activation

2020 ◽  
Author(s):  
Mahdieh Yazdani ◽  
Guohui Zhang ◽  
Zhiguang Jia ◽  
Jingyi Shi ◽  
Jianmin Cui ◽  
...  
Keyword(s):  
Protein Function ◽  
Bk Channels ◽  
Bk Channel ◽  
Atomistic Simulations ◽  
Allosteric Coupling ◽  
Linker Sequence ◽  
Sequence Scrambling ◽  
Intracellular Ca ◽  
Nonspecific Interactions ◽  
Potential Complex

Large-conductance potassium (BK) channels are transmembrane (TM) proteins that can be synergistically and independently activated by membrane voltage and intracellular Ca2+. The only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue "C-linker". To determine the linker's role in BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combing atomistic simulations and further mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results also suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus.

scholarly journals Phospho-islands and the evolution of phosphorylated amino acids in mammals

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10436
Author(s):  
Mikhail Moldovan ◽  
Mikhail S. Gelfand
Keyword(s):  
Amino Acids ◽  
Protein Function ◽  
Chemical Properties ◽  
Evolutionary Analysis ◽  
Post Translational Modification ◽  
Evolutionary Patterns ◽  
Charged Amino Acids ◽  
Physico Chemical ◽  
Frequent Mutations ◽  
Evolutionary Aspects

Background Protein phosphorylation is the best studied post-translational modification strongly influencing protein function. Phosphorylated amino acids not only differ in physico-chemical properties from non-phosphorylated counterparts, but also exhibit different evolutionary patterns, tending to mutate to and originate from negatively charged amino acids (NCAs). The distribution of phosphosites along protein sequences is non-uniform, as phosphosites tend to cluster, forming so-called phospho-islands. Methods Here, we have developed a hidden Markov model-based procedure for the identification of phospho-islands and studied the properties of the obtained phosphorylation clusters. To check robustness of evolutionary analysis, we consider different models for the reconstructions of ancestral phosphorylation states. Results Clustered phosphosites differ from individual phosphosites in several functional and evolutionary aspects including underrepresentation of phosphotyrosines, higher conservation, more frequent mutations to NCAs. The spectrum of tissues, frequencies of specific phosphorylation contexts, and mutational patterns observed near clustered sites also are different.

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