B1 Domain Of Protein Research Articles

NetSci: A Library for High Performance Biomolecular Simulation Network Analysis Computation.

We present the NetSci program-an open-source scientific software package designed for estimating mutual information (MI) between data sets using GPU acceleration and a k-nearest-neighbor algorithm. This approach significantly enhances calculation speed, achieving improvements of several orders of magnitude over traditional CPU-based methods, with data set size limits dictated only by available hardware. To validate NetSci, we accurately compute MI for an analytically verifiable two-dimensional Gaussian distribution and replicate the generalized correlation (GC) analysis previously conducted on the B1 domain of protein G. We also apply NetSci to molecular dynamics simulations of the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA) pump, exploring the allosteric mechanisms and pathways influenced by ATP and 2'-deoxy-ATP (dATP) binding. Our analysis reveals distinct allosteric effects induced by ATP compared to dATP, with predicted information pathways from the bound nucleotide to the calcium-binding domain differing based on the nucleotide involved. NetSci proves to be a valuable tool for estimating MI and GC in various data sets and is particularly effective for analyzing intraprotein communication and information transfer.

Characterizing Fast Conformational Exchange of Aromatic Rings Using Residual Dipolar Couplings: Distinguishing Jumplike Flips from Other Exchange Mechanisms.

Aromatic ring flips are a hallmark of protein dynamics. They are experimentally studied by NMR spectroscopy, where recent advances have led to improved characterization across a wide range of time scales. Results on different proteins have been interpreted as continuous diffusive ring rotations or jumplike flips, leading to diverging views of the protein interior as being fluidlike or solidlike, respectively. It is challenging to distinguish between these mechanisms and other types of conformational exchange because chemical-shift-mediated line broadening provides only conclusive evidence for ring flips only if the system can be moved from the slow- to intermediate/fast-exchange regime. Moreover, whenever the chemical shift difference between the two symmetry-related sites is close to zero, it is not generally possible to determine the exchange time scale. Here we resolve these issues by measuring residual dipolar coupling (RDC)-mediated exchange contributions using NMR relaxation dispersion experiments on proteins dissolved in dilute liquid crystalline media. Excellent agreement is found between the experimental difference in RDC between the two symmetry-related sites and the value calculated from high-resolution X-ray structures, demonstrating that dynamics measured for F52 in the B1 domain of protein G reports on distinct, jumplike flips rather than other types of conformational exchange.

An account on the factors determining the extra stability of the β–hairpin from B1 domain of protein G

The folding–unfolding of a 16 residue polypeptide, a β–hairpin in B1 domain of protein G is investigated here to account for the factors assisting the extra stability of the polypeptide in the presence of an explicit solvent and even when a denaturant like urea is present in the medium. It is observed here that the backbone H–bond network well defines the folded state and is even capable of forming the folded state, but it is not the only criteria for making a stable β–hairpin fold. Factors such as the side chain H–bonds and the alignment of the certain hydrophobic group side chains play a prominent role in preserving the β–hairpin structure and thus providing an extra stability to the hairpin architecture. It is also affirmed that the mentioned hydrophobic groups side chain interactions are very crucial in holding the β–hairpin together and without which the hairpin collapses completely. We also confirm that the denaturant urea acts on the GB1–hairpin backbone H–bonds and in the presence of strong hydrophobic interactions with a consistent side chain H–bonding network, the denaturation being comparatively a slower process with respect to the protein devoid of the side chain interactions. Communicated by Ramaswamy H. Sarma

Removing a single-arm species by Fibro PrismA in purifying an asymmetric IgG-like bispecific antibody

MabSelect PrismA is an affinity resin whose ligand is derived from the B-domain of Protein A. Compared to its predecessor resins (i.e., MabSelect SuRe LX and MabSelect SuRe), the PrismA ligand has enhanced affinity for the VH3 chain. Consistently, when being used for the purification of an asymmetric IgG-like bispecific antibody (bsAb), MabSelect PrismA resin shows certain resolution between the intact product and a single-arm byproduct, which contain the same Fc region but different numbers of VH3 domain. Fibro PrismA is a newly launched product which has the same PrismA Protein A ligand as MabSelect PrismA but uses cellulose fiber instead of resin as its matrix. It was learned that Fibro PrismA, in comparison to PrismA resin, exhibits further improved resolution, allowing efficient clearance of the single-arm byproduct as well as good recovery of the target bsAb. This finding suggests that Fibro PrismA provides a potential solution for separating antibody species containing the same Fc region but different numbers of VH3 domain, which can otherwise be difficult to separate.

The nature of persistent interactions in two model β-grasp proteins reveals the advantage of symmetry in stability.

Two proteins within the β-grasp superfamily, the B1-domain of protein G and the small archaeal modifier protein 1, were investigated to elucidate the key determinants of structural stability at the level of individual interactions. These symmetrical proteins both contain two β-hairpins which form a sheet flanked by a central α-helix. They were subjected to high temperature molecular dynamics simulations and the detailed behavior of each long-range interaction was characterized. The results revealed that in GB1 the most stable region was the C-terminal hairpin and in SAMP1 it was the opposite, the N-terminal hairpin. Experimental results for GB1 support this finding. In conclusion, it appears that the difference in the location and number of hydrophobic interactions dictate the differential stability which is accommodated due to structural symmetry of the β-grasp fold. Thus, the hairpins are interchangeable and in nature this lends itself to adaptability and flexibility.

Incorporating thioamides into proteins by native chemical ligation.

The thioamide is a versatile replacement of the peptide backbone with altered hydrogen bonding and conformational preferences, as well the ability participate in energy and electron transfer processes. Semi-synthetic incorporation of a thioamide into a protein can be used to study protein folding or protein/protein interactions using these properties. Semi-synthesis also provides the opportunity to study the role of thioamides in natural proteins. Here we outline the semi-synthesis of a model protein, the B1 domain of protein G (GB1) with a thioamide at the N-terminus or the C-terminus. The thioamide is synthetically incorporated into a fragment by solid-phase peptide synthesis, whereas the remainder of the protein is recombinantly expressed. Then, the two fragments are joined by native chemical ligation. The explicit protocol for GB1 synthesis is accompanied by examples of applications with GB1 and other proteins in structural biology and protein misfolding studies.

Inferring Protein Sequence-Function Relationships with Large-Scale Positive-Unlabeled Learning.

Machine learning can infer how protein sequence maps to function without requiring a detailed understanding of the underlying physical or biological mechanisms. It is challenging to apply existing supervised learning frameworks to large-scale experimental data generated by deep mutational scanning (DMS) and related methods. DMS data often contain high-dimensional and correlated sequence variables, experimental sampling error and bias, and the presence of missing data. Notably, most DMS data do not contain examples of negative sequences, making it challenging to directly estimate how sequence affects function. Here, we develop a positive-unlabeled (PU) learning framework to infer sequence-function relationships from large-scale DMS data. Our PU learning method displays excellent predictive performance across ten large-scale sequence-function datasets, representing proteins of different folds, functions, and library types. The estimated parameters pinpoint key residues that dictate protein structure and function. Finally, we apply our statistical sequence-function model to design highly stabilized enzymes.

HACANCOi: a new H\u03b1-detected experiment for backbone resonance assignment of intrinsically disordered proteins

Unidirectional coherence transfer is highly efficient in intrinsically disordered proteins (IDPs). Their elevated ps-ns timescale dynamics ensures long transverse (T2) relaxation times allowing sophisticated coherence transfer pathway selection in comparison to folded proteins. 1Hα-detection ensures non-susceptibility to chemical exchange with the solvent and enables chemical shift assignment of consecutive proline residues, typically abundant in IDPs. However, many IDPs undergo a disorder-to-order transition upon interaction with their target protein, which leads to the loss of the favorable relaxation properties. Long coherence transfer routes now result in prohibitively large decrease in sensitivity. We introduce a novel 4D 1Hα-detected experiment HACANCOi, together with its 3D implementation, which warrant high sensitivity for the assignment of proline-rich regions in IDPs in complex with a globular protein. The experiment correlates 1Hαi, 13Cαi, 15Ni and ^{13} C^{prime}_{i} spins by transferring the magnetization concomitantly from 13Cαi to 15Ni and ^{13} C^{prime}_{i}. The B1 domain of protein G (GB1), and the enteropathogenic E.coli EspF in complex with human SNX9 SH3, serve as model systems to demonstrate the attainable sensitivity and successful sequential assignment.

How well do force fields capture the strength of salt bridges in proteins?

Salt bridges form between pairs of ionisable residues in close proximity and are important interactions in proteins. While salt bridges are known to be important both for protein stability, recognition and regulation, we still do not have fully accurate predictive models to assess the energetic contributions of salt bridges. Molecular dynamics simulation is one technique that may be used study the complex relationship between structure, solvation and energetics of salt bridges, but the accuracy of such simulations depends on the force field used. We have used NMR data on the B1 domain of protein G (GB1) to benchmark molecular dynamics simulations. Using enhanced sampling simulations, we calculated the free energy of forming a salt bridge for three possible lysine-carboxylate ionic interactions in GB1. The NMR experiments showed that these interactions are either not formed, or only very weakly formed, in solution. In contrast, we show that the stability of the salt bridges is overestimated, to different extents, in simulations of GB1 using seven out of eight commonly used combinations of fixed charge force fields and water models. We also find that the Amber ff15ipq force field gives rise to weaker salt bridges in good agreement with the NMR experiments. We conclude that many force fields appear to overstabilize these ionic interactions, and that further work may be needed to refine our ability to model quantitatively the stability of salt bridges through simulations. We also suggest that comparisons between NMR experiments and simulations will play a crucial role in furthering our understanding of this important interaction.

Cotranslocational processing of the protein substrate calmodulin by an AAA+ unfoldase occurs via unfolding and refolding intermediates

Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments.

Quantification of size effect on protein rotational mobility in cells by 19F NMR spectroscopy.

Protein diffusion in living cells might differ significantly from that measured in vitro. Little is known about the effect of globular protein size on rotational diffusion in cells because each protein has distinct surface properties, which result in different interactions with cellular components. To overcome this problem, the B1 domain of protein G (GB1) and several concatemers of the protein were labeled with 5-fluorotryptophan and studied by 19F NMR in Escherichia coli cells, Xenopus laevis oocytes, and in aqueous solutions crowded with glycerol, or Ficoll70™ and lysozyme. Relaxation data show that the size dependence of protein rotation in cells is due to weak interactions of the target protein with cellular components, but the effect of these interactions decreases as protein size increases. The results provide valuable information for interpreting protein diffusion data acquired in living cells. Graphical abstract Size matters. The protein rotational mobility in living cells was assessed by 19F NMR. The size dependence effect may arise from weak interactions between protein and cytoplasmic components.

Using Hydrogen Bond Surrogate Technology to Stabilize Beta-Hairpins

Beta-hairpins are comprised of two β-strands connected by a short hairpin loop. The β-hairpin motif is critical in many protein folds and diverse protein-protein interactions (PPIs). Extensive studies on the forces governing β-hairpin folding and stability have led to the development of many strategies for β-hairpin stabilization, including side-chain cross-linking, rigid loop mimics, and macrocyclization. While these strategies have provided valuable insight into β-hairpin behavior, sacrifice of side chains may limit their application in the design and evaluation of β-hairpin PPI inhibitors. To overcome these challenges, we investigated the hydrogen bond surrogate (HBS) strategy as a method for β-hairpin stabilization. Previous studies have demonstrated the effectiveness of the HBS strategy in nucleation and stabilization of peptides in the α-helical conformation. Alpha-helix nucleation is accomplished by replacing the N-terminal backbone hydrogen bond with a covalent bond using ring-closing olefin metathesis on alkene substrates positioned at the i and i+4 peptide positions. Importantly, mimicry of a backbone hydrogen bond yields peptides with a full complement of side-chain functional groups. Our model system is a well-characterized β-hairpin derived from the B1 domain of protein G (GB1). The HBS macrocycle was formed on solid support by joining alkenes that mimic a hydrogen bond between the peptide's N- and C-termini. Conformational stabilities of unconstrained and HBS peptides were evaluated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Consistent with previous results, we find that the unconstrained peptides show weak propensity for the β-hairpin conformation while HBS macrocyclization significantly increases β-hairpin propensity. We envision that the HBS strategy will be generally effective in stabilizing diverse β-hairpin sequences.

Investigating the Scaling Behavior of Multidomain Proteins under Force using Single Molecule and Ensemble Force-Clamp Spectroscopy

A large number of proteins operating under force in vivo are segregated into multidomains, and integrate the unfolding of some of these domains into their mechanical response. Due to the vectorial nature of the force, these proteins are best studied with force spectroscopy techniques. Here we combine single molecule magnetic tweezers with a new technique capable of measuring protein hydrogels using constant force protocols. For our studies we engineer constructs with the B1 domain of protein L, a model alpha-beta protein from Peptostreptococcus magnus. Using magnetic tweezers and HaloTag covalent attachment, we measure the unfolding and refolding of protein L in various force and solutions regimes. At single molecule level, domain unfolding is measured as step-increments in end-to-end length. The measured extensions and unfolding rates as a function of force allow us to estimate the energy barrier between the folded and unfolded states to ∼12 kT and validate the of the use worm-like chain model to describe the step size as a function of force, as previously reported by J. Valle-Orero et al, BBRC 460 (2015). Crosslinking multidomain protein L constructs engineered with eight repeats results in protein hydrogels, which are a few mm long and experience mN forces while extending several microns. We find that hydrogels based on protein L show at constant force a non-exponential smooth response to force and the presence of chemical denaturants or osmolytes have a significant effect on their elasticity. We propose a 3D model to explain the measured behavior of these protein hydrogels which is based on the free energy of a single protein domain under force and which assumes a random orientation and placement of the molecules inside the gel. This model reproduces qualitatively the measured force-clamp response of protein hydrogels and is the first step toward understanding the transition from the probabilistic response of single molecules to the deterministic response that characterizes the functioning of tissues and organs.

Field dependent R1 and R2 relaxation in B3 domain of protein G (GB3)

Field dependent R1 and R2 relaxation in B3 domain of protein G (GB3)

Suppressing the epimerization of endothioamide peptides during Fmoc/t-Bu-based solid phase peptide synthesis.

Despite a number of intriguing utilities associated with thioamide-containing peptides and proteins in the context of biophysics, pharmacology and chemical biology, it has hitherto remained as one of the underexplored territories of peptidomimetics. The synthesis of long mono to multiply substituted endothioamide peptides is invariably accompanied with severe epimerization, oxoamide formation and various other undesired side reactions, resulting in messy product profiles. This has completely restrained their use as novel chemical tools for biological studies. During the chain elongation of an N-terminally located thioamide peptide using the Fmoc/t-Bu chemistry, it becomes vulnerable to the repetitive basic treatments as required for such chemistry. The incompatibility of thioamide moiety with bases as well as strong coupling reagents leads to epimerization as well as other side reactions due to its nucleophilicity, resulting in the loss of the stereochemical identity of the thioamidated amino acid residue. An easy-to-implement and efficient protocol to synthesize long (>10-mer) endothioamide peptides, significantly suppressing epimerization and other side reactions using 10% piperidine/dimethylformamide for 1 min, is reported herein. The novelty of the protocol is shown through the efficient synthesis of a number of 10-12-mer mono to multiply thioamide-substituted peptides with broad substrate scopes. The utility of the protocol in the context of protein engineering and chemical protein synthesis is also shown through the synthesis of a thioamide version of the 16-mer peptide from the B1 domain of protein G. Such a protocol to synthesize long endothioamide peptides would open up avenues toward engineering and accessing novel thiopeptide and thioprotein-based chemical tools, the synthesis of which had been a serious hurdle thus far. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Quantifying protein dynamics in the ps-ns time regime by NMR relaxation.

Both 15N chemical shift anisotropy (CSA) and sufficiently rapid exchange linebroadening transitions exhibit relaxation contributions that are proportional to the square of the magnetic field. Deconvoluting these contributions is further complicated by residue-dependent variations in protein amide 15N CSA values which have proven difficult to accurately measure. Exploiting recently reported improvements for the implementation of T1 and T1ρ experiments, field strength-dependent studies have been carried out on the B3 domain of protein G (GB3) as well as on the immunophilin FKBP12 and a H87V variant of that protein in which the major conformational exchange linebroadening transition is suppressed. By applying a zero frequency spectral density rescaling analysis to the relaxation data collected at magnetic fields from 500 to 900MHz 1H, differential residue-specific 15N CSA values have been obtained for GB3 which correlate with those derived from solid state and liquid crystalline NMR measurements to a level similar to the correlation among those previously reported studies. Application of this analysis protocol to FKBP12 demonstrated an efficient quantitation of both weak exchange linebroadening contributions and differential residue-specific 15N CSA values. Experimental access to such differential residue-specific 15N CSA values should significantly facilitate more accurate comparisons with molecular dynamics simulations of protein motion that occurs within the timeframe of global molecular tumbling.

Electrostatic Contributions to Protein Quinary Structure.

There are four well-known levels of protein structure: primary (amino acid sequence), secondary (helices, sheets and turns), tertiary (three-dimensional structure) and quaternary (specific protein-protein interactions). The fifth level remains largely undefined because characterization of quinary structure, the transient but essential macromolecular interactions that organize the crowded cellular interior, requires the measurement of equilibrium thermodynamic parameters in living cells. We have overcome this challenge by quantifying the pH-dependence of quinary interactions in living Escherichia coli cells using the B1 domain of protein G (GB1, 6.2 kDa). To accomplish this goal, we buffered the cellular interior and used NMR-detected amide proton exchange to quantify the free energy of unfolding in cells. At neutral pH, the unfolding free energy in cells is comparable to that in buffered solution. As the pH decreases, the increased number of attractive interactions between E. coli proteins and GB1 destabilizes the protein in cells relative to buffer alone. The data show that electrostatic interactions contribute to quinary structure.

Improved Side Chain Dynamics in MARTINI Simulations of Protein-Lipid Interfaces.

Specific interactions of protein side chains and lipid membranes regulate the localization, orientation, and activity of many peripheral proteins. Here, we introduce a modification of the coarse-grained MARTINI protein model, called 'side chain fix' (scFix), that was necessary and sufficient to correctly sample the side chain dynamics of β-strands in several globular proteins. When compared to μs long atomistic simulations or previous experimental findings, scFix MARTINI simulations reproduced all key interactions between the well-studied PLC-δ1 pleckstrin homology domain and a phosphatidylinositol-4,5-bisphosphate (PIP2) containing lipid membrane. Moreover, the extended runtime and higher sampling speed enabled the systematic mapping of the protein's rolling motion at the membrane, the identification of short-lived and stable binding orientations, as well as the verification and prediction of already known and of novel transient PIP2 binding sites. scFix also showed promise to maintain proper side chain orientation in other secondary structural motifs of the α-spectrin SH3 domain, the B1 domain of protein G, and the villin headpiece. This suggests that scFix improves on the predictive power of MARTINI simulations regarding protein-lipid and protein-ligand interactions.

Elucidating the Key Determinants of Structure, Folding, and Stability for the ( 4β+ α ) Conformation of the B1 Domain of Protein G Using Bioinformatics Approaches.

The B1 domain of protein G (GB1) is a small, 56 amino acid bacterial immunoglobulin-binding protein with a 4β+ α fold. Architecturally, it is composed of a two-layer sandwich consisting of a four-stranded β -sheet that packs against an α -helix. Using several bioinformatics approaches, we investigated which residues may be key determinants of this fold. We identified nine structurally conserved amino acids using a conservation analysis and propose they are critical to forming and stabilizing the fold. The nine conserved residues form a predominantly hydrophobic nucleus within the core of GB1. A network analysis of all the long-range interactions in the structure of GB1 in concert with a betweenness centrality analysis revealed the relative significance of each conserved amino acid residue based on the number and location of the interactions. This bioinformatics analysis provides an important foundation for the design and interpretation of both computational and experimental work which may be helpful in solving the protein folding problem.

Folding Dynamics of B1 Domain of Protein G using Single Molecule Force Spectroscopy

The B1 domain of protein G is a well-studied model system of protein folding that has a mixed secondary structure, high stability and relatively slow folding kinetics. However recent measurements on the sub-millisecond timescale show significant complexity before final folding step at ∼1ms which depends on how folding is observed. Since protein folding is still a poorly understood process, different denaturants have been put into experiment to better understand the folding process of various proteins. Applying force is one natural method for denaturing proteins in order to observe the process of refolding in detail, but the time resolution of standard instruments typically make investigation of fast folding proteins difficult. In this study, two alkyne-containing unnatural amino acid residues were added to both N and C termini of GB1 and connected via click chemistry to double stranded DNA handles. The protein-DNA chimeras were then suspended between a pair of polystyrene beads held in high-resolution dual optical traps and reversible folding/unfolding events were observed both during force-extension pulling/relaxation experiments as well as under constant force feedback conditions. Results presented here show that since GB1 structure undergoes rapid switching between intermediate states of varied extension, thus a two-state model is not appropriate for understanding full folding path of this domain.