Protein Loop Research Articles

The ubiquitous pyridoxal 5'-phosphate-binding protein is also an RNA-binding protein.

The pyridoxal 5'-phosphate binding protein (PLP-BP) is believed to play a crucial role in PLP homeostasis, which may explain why it is found in living organisms from all kingdoms. Escherichia coli YggS is the most studied homolog, but human PLP-BP has also attracted much attention because variants of this protein are responsible for a severe form of B6-responsive neonatal epilepsy. Yet, how PLP-BP is involved in PLP homeostasis, and thus what its actual function is in cellular metabolism, is entirely unknown. The present study shows that YggS binds RNA and that the strength of this interaction is modulated by PLP. A key role in RNA binding is clearly played by Lys137, an invariant residue located on a protein loop away from the PLP binding site, whose importance has been highlighted previously. The interaction with RNA is evidently conserved, since it is also observed with human PLP-BP. The RNA binding site, which is apparently located at the entrance of the PLP-binding site, is also evolutionarily conserved. It is therefore reasonable to assume that PLP, by defining the conformation of the protein, determines the RNA binding affinity. RNA-seq analysis of RNA co-purified with or captured by YggS revealed SsrA and RnpB RNAs, respectively involved in trans-translation and tRNA maturation, as the major molecular components. This work opens up new horizons for the function of the PLP-BP, which could be related to its interaction with RNA and modulated by PLP, and thus play a role in an as yet unknown regulatory mechanism.

The Trajectory of Damaged-Base Eversion into the Active Site of Apurinic/Apyrimidinic Endonuclease APE1 Regulates This Enzyme's Substrate Specificity.

Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for the hydrolysis of the phosphodiester bond on the 5' side of an apurinic/apyrimidinic site during base excision repair. Moreover, in DNA, this enzyme can recognize nucleotides containing such damaged bases as 5,6-dihydro-2'-deoxyuridine (DHU), 2'-deoxyuridine (dU), alpha-2'-deoxyadenosine (αA), and 1,N6-ethenoadenosine (εA). Previously, by pulsed electron-electron double resonance spectroscopy and pre-steady-state kinetic analysis, we have revealed multistep DNA rearrangements during the formation of the catalytic complex. In the present study, the modeling of the eversion trajectory of nucleotides with various damaged bases was performed by directed molecular dynamics simulations. It was found that each damaged base at the beginning of the eversion interacts with protein loop Val196-Arg201, which should be moved to enable further nucleotide eversion. This movement involves a shift in loop Val196-Arg201 away from loop Asn253-Thr257 and requires the disruption of contacts between these loops. The Glu260Ala substitution facilitates the separation of the two loops. Moreover, conformational changes in the Asn253-Thr257 loop should occur in the second half of the lesion eversion trajectory. All these perturbations within the protein globule tend to reduce steric interactions of each damaged base with the protein during the eversion of the nucleotide from DNA and movement to the active site. These perturbations are important determinants of substrate specificity of endonuclease APE1.

Mutations in the essential outer membrane protein BamA contribute to Escherichia coli resistance to the antimicrobial peptide TAT-RasGAP317-326

Antimicrobial peptides (AMPs) are promising alternatives to classical antibiotics against antibiotic-resistant pathogens. TAT-RasGAP317-326 is an AMP with broad range antibacterial activity, but its mechanism of action is unknown. In this study, we analysed a strain of E. coli with extensive resistance to TAT-RasGAP317-326 but not to other AMPs that we obtained after twenty passages during an in vitro resistance selection experiment. This strain accumulated four mutations. One of these is a point mutation in bamA, which encodes an essential protein involved in the folding and proper insertion of outer membrane proteins. The mutation resulted in a change of the charge in a surface-exposed negatively charged loop of the BamA protein. Using CRISPR-Cas9-based targeted mutagenesis, we showed that mutations lowering the negative charge of this loop decreased sensitivity of E. coli to TAT-RasGAP317-326. In silico simulations unveiled the molecular driving forces responsible for the interaction between TAT-RasGAP317-326 and BamA. These results indicated that electrostatic interactions, particularly hydrogen bonds, are involved in the stability of the molecular complex, representing a predictive fingerprint of the TAT-RasGAP317-326 - BamA interaction strength. Interestingly, BamA activity was only partially affected by TAT-RasGAP317-326, indicating that BamA may function as a specific receptor for this AMP. Our results indicate that binding and entry of TAT-RasGAP317-326 may involve different mechanisms compared to other AMPs, which is in line with limited cross-resistance observed between different AMPs. This limited cross-resistance is important for the clinical application of AMPs towards drug-resistant pathogens.

Discovery and computational exploration of SNPs in GNRHR gene and their influence on protein structure and function in Indian goat breeds

This study focuses on identifying functional single nucleotide polymorphisms (SNPs) within the gonadotropin-releasing hormone receptor (GNRHR) gene and conducting subsequent in-silico analysis of their effects on protein structure and function in two distinct South Indian goat breeds, namely Attapady Black (n = 120) and Malabari goats (n = 180), known for their divergent prolificacy traits. Utilizing a DNA pool sequencing assay, ten SNPs were uncovered in the study population: c.-1129T>G, c.-1069A>G, c.-978A>C, c.-605A>G, c.-33A>G, c.-29T>G, c.48G>A, c.75G>A, c.209T>G, and c.*212A>G. Notably, two polymorphisms, c.-1129T>G and c.-33A>G, were novel. Additionally, two polymorphisms, c.-33A>G and c.-978A>C, were exclusive to Malabari goats. Analysis of upstream variants revealed modifications to transcription factor and micro-RNA (miRNA) binding sites, suggesting potential alterations in GNRHR expression. Of particular significance was the non-synonymous exonic variant at c.209T>G locus, resulting in methionine to arginine substitution at the 70th position within the first intracellular loop of the receptor protein. This amino acid change may have implications for the functional dynamics of the receptor as GnRHR intracellular loops are involved in G protein coupling thereby facilitation of downstream signalling pathways. The identified SNPs and their in-silico impact analysis contribute to our understanding of the molecular mechanisms underlying reproductive traits in these goat populations, with implications for future breeding strategies and genomic selection programs.

Cryptic Extensibility in von Willebrand Factor Revealed by Molecular Nanodissection.

Von Willebrand factor (VWF) is a multimer with a variable number of protomers, each of which is a head-to-head dimer of two multi-domain monomers. VWF responds to shear through the unfolding and extension of distinct domains, thereby mediating platelet adhesion and aggregation to the injured blood vessel wall. VWF's C1-6 segment uncoils and then the A2 domain unfolds and extends in a hierarchical and sequential manner. However, it is unclear whether there is any reservoir of further extensibility. Here, we explored the presence of cryptic extensibility in VWF by nanodissecting individual, pre-stretched multimers with atomic force microscopy (AFM). The AFM cantilever tip was pressed into the surface and moved in a direction perpendicular to the VWF axis. It was possible to pull out protein loops from VWF, which resulted in a mean contour length gain of 217 nm. In some cases, the loop became cleaved, and a gap was present along the contour. Frequently, small nodules appeared in the loops, indicating that parts of the nanodissected VWF segment remained folded. After analyzing the nodal structure, we conclude that the cryptic extensibility lies within the C1-6 and A1-3 regions. Cryptic extensibility may play a role in maintaining VWF's functionality in extreme shear conditions.

Are Viruses Taxonomic Units? A Protein Domain and Loop-Centric Phylogenomic Assessment.

Virus taxonomy uses a Linnaean-like subsumption hierarchy to classify viruses into taxonomic units at species and higher rank levels. Virus species are considered monophyletic groups of mobile genetic elements (MGEs) often delimited by the phylogenetic analysis of aligned genomic or metagenomic sequences. Taxonomic units are assumed to be independent organizational, functional and evolutionary units that follow a 'natural history' rationale. Here, I use phylogenomic and other arguments to show that viruses are not self-standing genetically-driven systems acting as evolutionary units. Instead, they are crucial components of holobionts, which are units of biological organization that dynamically integrate the genetics, epigenetic, physiological and functional properties of their co-evolving members. Remarkably, phylogenomic analyses show that viruses share protein domains and loops with cells throughout history via massive processes of reticulate evolution, helping spread evolutionary innovations across a wider taxonomic spectrum. Thus, viruses are not merely MGEs or microbes. Instead, their genomes and proteomes conduct cellularly integrated processes akin to those cataloged by the GO Consortium. This prompts the generation of compositional hierarchies that replace the 'is-a-kind-of' by a 'is-a-part-of' logic to better describe the mereology of integrated cellular and viral makeup. My analysis demands a new paradigm that integrates virus taxonomy into a modern evolutionarily centered taxonomy of organisms.

Severe Combined Immunodeficiency from a Homozygous DNA Ligase 1 Mutant with Reduced Catalytic Activity but Increased Ligation Fidelity

A cell’s ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3’ end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.

Highly Accurate and Efficient Deep Learning Paradigm for Full-Atom Protein Loop Modeling with KarmaLoop.

Protein loop modeling is a challenging yet highly nontrivial task in protein structure prediction. Despite recent progress, existing methods including knowledge-based, abinitio, hybrid, and deep learning (DL) methods fall substantially short of either atomic accuracy or computational efficiency. To overcome these limitations, we present KarmaLoop, a novel paradigm that distinguishes itself as the first DL method centered on full-atom (encompassing both backbone and side-chain heavy atoms) protein loop modeling. Our results demonstrate that KarmaLoop considerably outperforms conventional and DL-based methods of loop modeling in terms of both accuracy and efficiency, with the average RMSDs of 1.77 and 1.95 Å for the CASP13+14 and CASP15 benchmark datasets, respectively, and manifests at least 2 orders of magnitude speedup in general compared with other methods. Consequently, our comprehensive evaluations indicate that KarmaLoop provides a state-of-the-art DL solution for protein loop modeling, with the potential to hasten the advancement of protein engineering, antibody-antigen recognition, and drug design.

An alternative conformation of the N-terminal loop of human dihydroorotate dehydrogenase drives binding to a potent antiproliferative agent.

Over the years, human dihydroorotate dehydrogenase (hDHODH), which is a key player in the de novo pyrimidine-biosynthesis pathway, has been targeted in the treatment of several conditions, including autoimmune disorders and acute myelogenous leukaemia, as well as in host-targeted antiviral therapy. A molecular exploration of its inhibitor-binding behaviours yielded promising candidates for innovative drug design. A detailed description of the enzymatic pharmacophore drove the decoration of well-established inhibitory scaffolds, thus gaining further in vitro and in vivo efficacy. In the present work, using X-ray crystallography, an atypical rearrangement was identified in the binding pose of a potent inhibitor characterized by a polar pyridine-based moiety (compound 18). The crystal structure shows that upon binding compound 18 the dynamics of a protein loop involved in a gating mechanism at the cofactor-binding site is modulated by the presence of three water molecules, thus fine-tuning the polarity/hydrophobicity of the binding pocket. These solvent molecules are engaged in the formation of a hydrogen-bond mesh in which one of them establishes a direct contact with the pyridine moiety of compound 18, thus paving the way for a reappraisal of the inhibition of hDHODH. Using an integrated approach, the thermodynamics of such a modulation is described by means of isothermal titration calorimetry coupled with molecular modelling. These structural insights will guide future drug design to obtain a finer Kd/logD7.4 balance and identify membrane-permeable molecules with a drug-like profile in terms of water solubility.

Probability Density Reweighting of High-Temperature Molecular Dynamics.

Molecular dynamics (MD) simulation is a popular method for elucidating the structures and functions of biomolecules. However, exploring the conformational space, especially for large systems with slow transitions, often requires enhanced sampling methods. Although conducting MD at high temperatures provides a straightforward approach, resulting conformational ensembles diverge significantly from those at low temperatures. To address this discrepancy, we propose a novel probability density-based reweighting (PDR) method. PDR exhibits robust performance across four distinct systems, including a miniprotein, a cyclic peptide, a protein loop, and a protein-peptide complex. It accurately restores the conformational distributions at high temperatures to those at low temperatures. Additionally, we apply PDR to reweight previously studied high-T MD simulations of 12 protein-peptide complexes, enabling a comprehensive investigation of the conformational space of protein-peptide complexes.

Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Lineage Replacement and Genetic Changes of Four HR-HPV Types during the Period of Vaccine Coverage: A Six-Year Retrospective Study in Eastern China.

This study aimed to provide clinical evidence for lineage replacement and genetic changes of High-Risk Human Papillomavirus (HR-HPV) during the period of vaccine coverage and characterize those changes in eastern China. This study consisted of two stages. A total of 90,583 patients visiting the Obstetrics and Gynecology Hospital of Fudan University from March 2018 to March 2022 were included in the HPV typing analysis. Another 1076 patients who tested positive for HPV31, 33, 52, or 58 from November 2020 to August 2023 were further included for HPV sequencing. Vaccination records, especially vaccine types and the third dose administration time, medical history, and cervical cytology samples were collected. Viral DNA sequencing was then conducted, followed by phylogenetic analysis and sequence alignment. The overall proportion of HPV31 and 58 infections increased by 1.23% and 0.51%, respectively, while infection by HPV33 and 52 decreased by 0.42% and 1.43%, respectively, within the four-year vaccination coverage period. The proportion of HPV31 C lineage infections showed a 22.17% increase in the vaccinated group, while that of the HPV58 A2 sublineage showed a 12.96% increase. T267A and T274N in the F-G loop of HPV31 L1 protein, L150F in the D-E loop, and T375N in the H-I loop of HPV58 L1 protein were identified as high-frequency escape-related mutations. Differences in epidemic lineage changes and dominant mutation accumulation may result in a proportional difference in trends of HPV infection. New epidemic lineages and high-frequency escape-related mutations should be noted during the vaccine coverage period, and regional epidemic variants should be considered during the development of next-generation vaccines.

Characterization of Two Highly Specific Monoclonal Antibodies Targeting the Glycan Loop of the Zika Virus Envelope Protein.

Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.

Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon.

Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.

Epitope(s) involving amino acids of the fusion loop of Japanese encephalitis virus envelope protein is(are) important to elicit protective immunity.

Introduction of mutations into the fusion loop is one potential strategy for generating safe dengue and Zika vaccines by reducing the risk of severe dengue following subsequent infections, and for constructing live-attenuated vaccine candidates against newly emerging Japanese encephalitis virus (JEV) or Japanese encephalitis (JE) serocomplex virus. The monoclonal antibody studies indicated the fusion loop of JE serocomplex viruses primarily comprised non-neutralizing epitopes. However, the present study demonstrates that the JEV fusion loop plays a critical role in eliciting protective immunity in mice. Modifications to the fusion loop of JE serocomplex viruses might negatively affect vaccine efficacy compared to dengue and zika serocomplex viruses. Further studies are required to assess the impact of mutant fusion loop encoded by commonly used JEV vaccine strains on vaccine efficacy or safety after subsequent dengue virus infection.

Activation and friction in enzymatic loop opening and closing dynamics

Protein loop dynamics have recently been recognized as central to enzymatic activity, specificity and stability. However, the factors controlling loop opening and closing kinetics have remained elusive. Here, we combine molecular dynamics simulations with string-method determination of complex reaction coordinates to elucidate the molecular mechanism and rate-limiting step for WPD-loop dynamics in the PTP1B enzyme. While protein conformational dynamics is often represented as diffusive motion hindered by solvent viscosity and internal friction, we demonstrate that loop opening and closing is activated. It is governed by torsional rearrangement around a single loop peptide group and by significant friction caused by backbone adjustments, which can dynamically trap the loop. Considering both torsional barrier and time-dependent friction, our calculated rate constants exhibit very good agreement with experimental measurements, reproducing the change in loop opening kinetics between proteins. Furthermore, we demonstrate the applicability of our results to other enzymatic loops, including the M20 DHFR loop, thereby offering prospects for loop engineering potentially leading to enhanced designs.

Designed allosteric protein logic.

The regulation of protein function by external or internal signals is one of the key features of living organisms. The ability to directly control the function of a selected protein would represent a valuable tool for regulating biological processes. Here, we present a generally applicable regulation of proteins called INSRTR, based on inserting a peptide into a loop of a target protein that retains its function. We demonstrate the versatility and robustness of coiled-coil-mediated regulation, which enables designs for either inactivation or activation of selected protein functions, and implementation of two-input logic functions with rapid response in mammalian cells. The selection of insertion positions in tested proteins was facilitated by using a predictive machine learning model. We showcase the robustness of the INSRTR strategy on proteins with diverse folds and biological functions, including enzymes, signaling mediators, DNA binders, transcriptional regulators, reporters, and antibody domains implemented as chimeric antigen receptors in T cells. Our findings highlight the potential of INSRTR as a powerful tool for precise control of protein function, advancing our understanding of biological processes and developing biotechnological and therapeutic interventions.

Protein-lipid interactions and protein anchoring modulate the modes of association of the globular domain of the Prion protein and Doppel protein to model membrane patches.

The Prion protein is the molecular hallmark of the incurable prion diseases affecting mammals, including humans. The protein-only hypothesis states that the misfolding, accumulation, and deposition of the Prion protein play a critical role in toxicity. The cellular Prion protein (PrPC) anchors to the extracellular leaflet of the plasma membrane and prefers cholesterol- and sphingomyelin-rich membrane domains. Conformational Prion protein conversion into the pathological isoform happens on the cell surface. In vitro and in vivo experiments indicate that Prion protein misfolding, aggregation, and toxicity are sensitive to the lipid composition of plasma membranes and vesicles. A picture of the underlying biophysical driving forces that explain the effect of Prion protein - lipid interactions in physiological conditions is needed to develop a structural model of Prion protein conformational conversion. To this end, we use molecular dynamics simulations that mimic the interactions between the globular domain of PrPC anchored to model membrane patches. In addition, we also simulate the Doppel protein anchored to such membrane patches. The Doppel protein is the closest in the phylogenetic tree to PrPC, localizes in an extracellular milieu similar to that of PrPC, and exhibits a similar topology to PrPC even if the amino acid sequence is only 25% identical. Our simulations show that specific protein-lipid interactions and conformational constraints imposed by GPI anchoring together favor specific binding sites in globular PrPC but not in Doppel. Interestingly, the binding sites we found in PrPC correspond to prion protein loops, which are critical in aggregation and prion disease transmission barrier (β2-α2 loop) and in initial spontaneous misfolding (α2-α3 loop). We also found that the membrane re-arranges locally to accommodate protein residues inserted in the membrane surface as a response to protein binding.

Assessing immune factors in maternal milk and paired infant plasma antibody binding to human rhinoviruses.

Before they can produce their own antibodies, newborns are protected from infections by transplacental transfer of maternal IgG antibodies and after birth through breast milk IgA antibodies. Rhinovirus (RV) infections are extremely common in early childhood, and while RV infections often result in only mild upper respiratory illnesses, they can also cause severe lower respiratory illnesses such as bronchiolitis and pneumonia. We used high-density peptide arrays to profile infant and maternal antibody reactivity to capsid and full proteome sequences of three human RVs - A16, B52, and C11. Numerous plasma IgG and breast milk IgA RV epitopes were identified that localized to regions of the RV capsid surface and interior, and also to several non-structural proteins. While most epitopes were bound by both IgG and IgA, there were several instances where isotype-specific and RV-specific binding were observed. We also profiled 62 unique RV-C protein loop sequences characteristic of this species' capsid VP1 protein. Many of the RV-C loop sequences were highly bound by IgG from one-year-old infants, indicating recent or ongoing active infections, or alternatively, a level of cross-reactivity among homologous RV-C sites.

LoopGrafter: Visual Support for the Grafting Workflow of Protein Loops.

In the process of understanding and redesigning the function of proteins in modern biochemistry, protein engineers are increasingly focusing on the exploration of regions in proteins called loops. Analyzing various characteristics of these regions helps the experts to design the transfer of the desired function from one protein to another. This process is denoted as loop grafting. As this process requires extensive manual treatment and currently there is no proper visual support for it, we designed LoopGrafter: a web-based tool that provides experts with visual support through all the loop grafting pipeline steps. The tool is logically divided into several phases, starting with the definition of two input proteins and ending with a set of grafted proteins. Each phase is supported by a specific set of abstracted 2D visual representations of loaded proteins and their loops that are interactively linked with the 3D view onto proteins. By sequentially passing through the individual phases, the user is shaping the list of loops that are potential candidates for loop grafting. In the end, the actual in-silico insertion of the loop candidates from one protein to the other is performed and the results are visually presented to the user. In this way, the fully computational rational design of proteins and their loops results in newly designed protein structures that can be further assembled and tested through in-vitro experiments. LoopGrafter was designed in tight collaboration with protein engineers, and its final appearance reflects many testing iterations. We showcase the contribution of LoopGrafter on a real case scenario and provide the readers with the experts' feedback, confirming the usefulness of our tool.