Biology subjects

Suresh Kumar, T. K.

Publications and source records attributed to Suresh Kumar, T. K..

5 recordsLinked to original sources

bioRxiv2022

cpSRP43 is both highly flexible and stable: Structural insights using a combined experimental and computational approach

The novel multidomain protein, cpSRP43, is a unique subunit of the post-translational chloroplast signal recognition particle (cpSRP) targeting pathway in higher plants. The cpSRP pathway is responsible for targeting and insertion of light-harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. Nuclear-encoded LHCPs are synthesized in the cytoplasm then imported into the chloroplast. Upon emergence into the stroma, LHCPs form a soluble transit complex with the cpSRP heterodimer, which is composed of cpSRP43 and cpSRP54, a 54 kDa subunit homologous to the universally conserved GTPase in cytosolic SRP pathways. cpSRP43 is irreplaceable as a chaperone to LHCPs in their translocation to the thylakoid membrane and remarkable in its ability to dissolve aggregates of LHCPs without the need for external energy input. In previous studies, cpSRP43 has demonstrated significant flexibility and interdomain dynamics. However, the high flexibility and structural dynamics of cpSRP43 is yet unexplained by current crystal structures of cpSRP43. This is due, in part, to the fact that free full length cpSRP43 is so flexible that it is unable to crystalize. In this study, we explore the structural stability of cpSRP43 under different conditions using various biophysical techniques and find that this protein is concurrently highly stable and flexible. This conclusion is interesting considering that stable proteins typically possess a non-dynamic structure. Molecular dynamics (MD) simulations which correlated with data from biophysical experimentation were used to explain the basis of the extraordinary stability of cpSRP43. This combination of biophysical data and microsecond-level MD simulations allows us to obtain a detailed perspective of the conformational landscape of these proteins.

biophysics↗

bioRxiv2021

Binding Affinity Estimation From Restrained Umbrella Sampling Simulations

The protein-ligand binding affinity quantifies the binding strength between a protein and its ligand. Computer modeling and simulations can be used to estimate the binding affinity or binding free energy using data- or physics-driven methods or a combination thereof. Here, we discuss a purely physics-based sampling approach based on biased molecular dynamics (MD) simulations, which in spirit is similar to the stratification strategy suggested previously by Woo and Roux. The proposed methodology uses umbrella sampling (US) simulations with additional restraints based on collective variables such as the orientation of the ligand. The novel extension of this strategy presented here uses a simplified and more general scheme that can be easily tailored for any system of interest. We estimate the binding affinity of human fibroblast growth factor 1 (hFGF1) to heparin hexasaccharide based on the available crystal structure of the complex as the initial model and four different variations of the proposed method to compare against the experimentally determined binding affinity obtained from isothermal calorimetry (ITC) experiments. Our results indicate that enhanced sampling methods that sample along the ligand-protein distance without restraining other degrees of freedom do not perform as well as those with additional restraint. In particular, restraining the orientation of the ligands plays a crucial role in reaching a reasonable estimate for binding affinity. The general framework presented here provides a flexible scheme for designing practical binding free energy estimation methods.

biophysics↗

bioRxiv2021

Stability Comparisons between Natural versus Engineered Archaeal Heat Shock Proteins

Crenarchaeal group II chaperonins (a.k.a., "heat shock" proteins, HSPs) are abundantly expressed in species of the family Sulfolobaceae. HSP and HSP{beta} expression is upregulated during thermal shock. HSPs are subunits of larger octadecameric complexes that function to protect intracellular proteins during thermal stress. Engineered HSPs have been constructed with the idea of protecting enzymes in industrial reactions. HSP{beta}-coh, a fusion protein comprised of HSP{beta} and type 1 cohesin from Clostridium thermocellum was used for proof-of-concept. Dockerin-endowed cellulolytic enzymes bind to the complex via cohesin-dockerin interactions. Enzymatic activity (i.e., hydrolysis of lignocellulose) is retained when the platform is used at high temperatures (e.g., 85-88{degrees}C). Moreover, enhancement persists on acid-pretreated substrates prompting the question: Are HSPs acid tolerant? In this study, HSP structural integrity is examined at different temperatures and pH. Far-UV circular dichroism and intrinsic fluorescence indicate HSP and HSP{beta} retain structural integrity at neutral pH over a range of temperatures (25-90{degrees}C) while HSP{beta}-coh is less tolerant to thermal stress. Structural integrity is compromised for all subunits at ultra-low pH (i.e., pH 2) with HSP showing the most susceptibility. Secondary structures of all HSPs are resilient under mildly acidic conditions (pH 4). ANS binding assays indicate a shift in tertiary structure for all subunits at ultra-low pH. Limited trypsin digestion reveals that the backbone of HSP{beta}-coh is the most flexible and HSP{beta} is the most resistant. Results suggest that HSP and HSP{beta} are more resilient than HSP{beta}-coh under thermal challenge and that there are limits to the acid tolerance of all HSPs.

biochemistry↗

bioRxiv2020

Mechanistic Picture for Monomeric Human Fibroblast Growth Factor 1 Stabilization by Heparin Binding

Human fibroblast growth factor (FGF) 1 or hFGF1 is a member of the FGF family that is involved in various vital processes such as cell proliferation, cell differentiation, angiogenesis and wound healing. hFGF1, which is associated with low stability in vivo, is known to be stabilized by binding heparin sulfate, a glycosaminoglycan that aids the protein in the activation of its cell surface receptor. The poor thermal and proteolytic stability of hFGF1 and the stabilizing role of heparin have long been observed experimentally; however, the mechanistic details of these phenomena are not well understood. Here, we have used a combination of microsecond-level equilibrium molecular dynamics (MD) simulations, and state-of-the-art enhanced sampling MD simulations to quantitatively characterize the structural dynamics of monomeric hFGF1 in the presence and absence of heparin hexasaccharide. We have observed a conformational change in the heparin-binding pocket of hFGF1 that occurs only in the absence of heparin. Several intramolecular hydrogen bonds were also identified within the heparin-binding pocket, that form only when hFGF1 interacts with heparin. The loss of both intermolecular and intramolecular electrostatic interactions in the absence of heparin plausibly leads to the observed conformational change. This conformational transition results in increased flexibility of the heparin-binding pocket and provides an explanation for the susceptibility of apo hFGF1 to proteolytic degradation and thermal instability. The hFGF1-heparin interaction has also been quantified using absolute binding free energy calculations. Binding affinity (Kd) estimates determined computationally using our novel MD approach are in good quantitative agreement with experimental Kd values from isothermal titration calorimetry experiments. The successful application of a combination of microsecond-level MD and accurate free energy calculations to explain the heparin-mediated stabilization of hFGF1 at a quantitative level, represents a promising approach for studying complex biomolecular interactions between proteins and their binding partners at a detailed molecular level using rigorous physics-based simulation techniques.

biophysics↗

bioRxiv2019

Chloroplast SRP43 Subunit Prevents Aggregation of Proteins

Integration of light-harvesting chlorophyll binding proteins into the thylakoid membrane requires a specific chaperone, being the cpSRP43 subunit, of the signal recognition particle pathway in chloroplasts. cpSRP43, unique to the chloroplast, is responsible for transport of LHCPs through the stroma as well as assisting in the correct folding, assembly and disaggregation of these proteins for the acquisition of light energy. cpSRP43 is a highly flexible, multidomain protein capable of binding distinct partners in the cpSRP pathway. cpSRP43 is an irreplaceable component, necessary for the accurate and successful integration of LHCPs. It can act as a disaggregase without any input of external energy. Its action is based on the ability to associate with variable regions of different proteins owing to the domains and flexibility within its distinctive structure. Understanding the unique capabilities of cpSRP43 in the chloroplast begs the question of its usefulness outside of the plant cell, as well as its yet unknown roles still within the plant cell. Although the capabilities of cpSRP43 as a hub protein, adept to binding many unknown partners, has been alluded to in other works, it has yet to be thoroughly investigated. In this study we discover that cpSRP43 can act as a generic chaperone for proteins other than LHCP/not native to the chloroplast. The high thermal stability of cpSRP43 has been demonstrated in the previous chapter by its ability to retain its secondary structure as well as withstand aggregation upon heating and cooling cycles as confirmed by absorbance, intrinsic tryptophan fluorescence and far UV circular dichroism spectroscopy. This property gives cpSRP43 the basis to act as a generic chaperone and provide protection like that of typical heat shock proteins. Carbonic anhydrase, Concanavalin A and hFGF1 (acidic human fibroblast growth factor), were selected as candidates for chaperoning activity by cpSRP43. In all three cases, heat-induced aggregation of the candidate protein was either eliminated or significantly reduced in the presence of cpSRP43. In the case of hFGF1, the bioactivity was preserved after heat-treatment in the presence of cpSRP43. We have proposed a mechanism by which cpSRP43 is able to execute this action however further investigation is warranted to determine the exact mechanism(s) which may vary dependent on the target protein.

biochemistry↗