A Thermodynamic Model for Interpreting Tryptophan Excitation-Energy-Dependent Fluorescence Spectra Provides Insight Into Protein Conformational Sampling and Stability

It is now over 30 years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. While there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the tryptophan REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides, to multitryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibodies in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating biomolecular simulation with experimental data to yield novel insights.

Mechanism of Laccase-assisted Tyrosine Grafting on Keratin using BSA as a Model Protein

Commercial hair perming uses strong reducing agents and is harmful to hair fiber’s quality even human health. In this study, tyrosine is adopted as a cross-linking agent between thiols as the shape-changing of hair involves breakage of disulfide bonds and the rearrangement of new bonds between keratin molecules. To investigate the mechanism of the cross-linking, bovine serum albumin (BSA) is used as a model protein. Molecular dynamics simulations give an insight on Cys solvent accessibility and protein stability for the wild type BSA and a designed BSA presenting the three broken disulfide bonds. A new cross-linked peptide, NECFLSHK-Tyrosine-Tyrosine-GACLLPK, inter- or intra- BSA monomers is formed, whose reactive cysteine residues are Cys-101 and Cys-176. Moreover, curling of Asian hair is conducted using tyrosine as a perming agent by laccase-assisted reaction. The optimized operational conditions are hair with cysteine pre-treatment (50.0 mM) followed by grafting with 3.0 mM tyrosine. The reshaped hair performed a better perming performance than commercial perming product before washing, although a lower perming efficiency after washing, however without strength loss and could be easily accomplished with a blow-drier. Hence, this new methodology may lead to the development of a gentle and user-friendly approach in the hair care industry.

Constitutive signal bias mediated by the human GHRHR splice variant 1

Alternative splicing of G protein–coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone–releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to β-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus β-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

A thermodynamic model for interpreting tryptophan excitation-energy-dependent fluorescence spectra provides insight into protein conformational sampling and stability

ABSTRACTIt is now over thirty years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. Whilst there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the protein REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides to multi-tryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibody in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating bimolecular simulation with experimental data to yield novel insights.

Constitutive signal bias mediated by the human GHRHR splice variant 1

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to β-arrestins. Based on the cryo-electron microscopy structures of SV1 in the apo state or in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. β-arrestins. Suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias towards β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Biophysical and structural studies reveal marginal stability of Acyl ACP Reductase: a crucial hydrocarbon biosynthetic enzyme

Background: Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in E. coli, leading to varying alkane levels. Intriguingly, the structural characterization remains largely elusive as AAR alone failed to form stable crystals, possibly due to a number of intrinsically flexible random regions. The present work attempts to fill a gap in the literature by investigating the crucial structural aspects of AAR protein associated with its stability and folding. Results: The AAR protein was recombinant expressed in E. coli and purified by metal affinity and gel filtration chromatography. Characterization by dynamic light scattering experiment revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles in the range of 36.4 to 51.6 nm. Intact mass spectrometry revealed that recombinant AAR was heterogenous due to diverse lipidation and de-lipidation resulted in a single mass peak of 40296.87 Da as predicted. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution. Lower concentration of GdnHCl appeared to be stabilizing the protein, suggesting that the protein milieu plays a significant role in dictating it’s folding. Standard free energy (∆GH2ONU) of ~4.5 kcal/mol for steady-state unfolding of AAR indicated borderline stability of the protein. Molecular dynamics simulation conducted on AAR structure in presence of KCl, an ionic solvent with similar properties as GdnHCl at lower concentrations, suggested that KCl mediates structural stabilization especially at the concentration of 375 mM, and thus was responsible for enhancing its activity. KCl presence also resulted in regional alteration towards the binding site of its neighbouring pathway enzyme, ADO, thus paving the way for coordinated catalysis.Conclusion: Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show path for building superior biocatalyst for higher biofuel production.

Finding Pathogenic nsSNP’s and their structural effect on COPS2 using Molecular Dynamic Approach

COP9 Signalosome Subunit 2 is a highly conserved multiprotein complex which is involved in the cellular process and developmental process. It is one of the essential components in the COP9 Signalosome Complex (CSN). It is also involved in neuronal differentiation interacting with NIF3L1. The gene involved in neuronal differentiation is negatively regulated due to the transcription co-repressor interaction of NIF3L1 with COPS2. In the present study, we have evaluated the outcome for 90 non-synonymous single nucleotide polymorphisms (nsSNP’s) in COPS2 gene through computational tools. After the analysis, 4 SNP’s (S120C, N144S, Y159H, R173C) were found to be deleterious. The native and mutated structures were prepared using discovery studio and docked to check the interactions with NIF3L1.On the basis of ZDOCK score the top 3 mutations (N144S, Y159H, R173C) were screened out. Further to analyze the effect of amino acid substitution on the molecular structure of protein Molecular Dynamics simulation was carried out. Analysis based on RMSD, RMSF, RG, H-bond showed a significant deviation in the graph, which demonstrated conformation change and instability compared to the wild structure. As it is known mutations in COPS2 gene can disrupt the normal activity of the CSN2 protein which may cause neuronal differentiation. Our results showed N144S, Y159H and R173C mutations are to be more pathogenic and may cause disease