Peptide Hormone Binding Protein Design
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Questions and Answers

What are peptide hormones known to adopt upon binding their receptors?

  • α-helical structures (correct)
  • Non-helical structures
  • Random coils
  • β-sheets

    • The RFdiffusion method can only generate binders with high affinity for structured proteins.

    False

    What is the main challenge in designing interactions between proteins and short peptides?

    The design of interactions is an unmet challenge due to their helical propensity.

    Peptide hormones, such as parathyroid hormone (PTH) and neuropeptide Y (NPY), play key roles in _____ biology.

    <p>human</p> Signup and view all the answers

    Match the peptide hormone with its correct description:

    <p>PTH = Regulates calcium levels NPY = Involved in appetite regulation GCG = Increases blood sugar levels SCT = Stimulates bile secretion</p> Signup and view all the answers

    Which method is mentioned for the sensitive detection of peptide hormones?

    <p>Mass spectrometry</p> Signup and view all the answers

    Antibodies are considered highly effective for quantifying all types of peptide hormones.

    <p>False</p> Signup and view all the answers

    What is a disadvantage of using antibodies for peptide hormone detection?

    <p>They require substantial resources to generate and often have less-than-desirable stability and reproducibility.</p> Signup and view all the answers

    What is a challenge in designing proteins that bind helical peptides?

    <p>Peptides are often unstructured in isolation</p> Signup and view all the answers

    The design of proteins that bind folded proteins is less challenging than that of those binding helical peptides.

    <p>True</p> Signup and view all the answers

    Name one method used for designing proteins that bind helical peptides.

    <p>Parametric generation or deep learning methods</p> Signup and view all the answers

    Peptides with _____ residues are often challenging to design binding proteins for due to their flexibility.

    <p>fewer</p> Signup and view all the answers

    What principle has been applied to design binders for calmodulin peptides?

    <p>Coiled-coil assemblies</p> Signup and view all the answers

    Hydrophobic surfaces in proteins increase the binding affinity for target peptides.

    <p>False</p> Signup and view all the answers

    What are the internal interactions of α-helical peptides primarily composed of?

    <p>Backbone-hydrogen bonding</p> Signup and view all the answers

    The binding affinity of designed proteins to targets was measured using a _____ assay.

    <p>NanoBiT split-luciferase</p> Signup and view all the answers

    Which method was shown to rapidly generate tight affinity binders with picomolar Kd values?

    <p>RFdiffusion</p> Signup and view all the answers

    The Hallucination approach requires pre-specification of the bound structures to generate high-affinity binders.

    <p>False</p> Signup and view all the answers

    Match the following peptide binding approaches with their descriptions:

    <p>Parametric generation = Sampling designed scaffolds with specified shapes Deep learning = Exploring sequences without pre-specified geometries Hallucination methods = Optimizing sequences for desired structural properties Threading approach = Adapting an existing structure to fit a new target</p> Signup and view all the answers

    Which of the following peptide binding targets was mentioned in the content?

    <p>Gastric inhibitory peptide</p> Signup and view all the answers

    What key strategy was used to achieve a more uniform distribution of supercoiling in the scaffold library?

    <p>biased sampling strategy</p> Signup and view all the answers

    High affinity for designed proteins was achieved in all peptide binding cases mentioned.

    <p>False</p> Signup and view all the answers

    RFjoint designs were validated and filtered in silico by _____ with an initial guess.

    <p>AF2</p> Signup and view all the answers

    What characterizes the proteins designed to bind helical peptides?

    <p>Helical bundles with an open groove</p> Signup and view all the answers

    Match the following methods with their applications:

    <p>RFjoint = Designing diversity in peptide binders RFdiffusion = Generating tight affinity binders Hallucination = Creating binders without pre-specification Rosetta = Filtering and scoring designs</p> Signup and view all the answers

    _____ measures the predicted alignment error across the interface in the Hallucination methods.

    <p>pAE</p> Signup and view all the answers

    What kind of library was sampled using a random sampling approach?

    <p>Parametric groove-shaped scaffold library</p> Signup and view all the answers

    Which of the following factors complicates the design of peptide-binding proteins?

    <p>Limited interaction sites on peptides</p> Signup and view all the answers

    Heterogeneous amino acid selections were achieved by independent random selections of helix distances.

    <p>True</p> Signup and view all the answers

    The method of sampling scaffolds allows for exploring variations in target binding designs.

    <p>True</p> Signup and view all the answers

    What specific method was used to extend the binding interface of the putative binder?

    <p>RFjoint Inpainting network</p> Signup and view all the answers

    The RFdiffusion method improves upon starting designs by partial ______ and denoising.

    <p>noising</p> Signup and view all the answers

    Which proteins were used as input for the RFjoint design?

    <p>PTH, GCG and NPY</p> Signup and view all the answers

    The Rosetta-designed binders were filtered on metrics that included buried polar surface area per residue.

    <p>False</p> Signup and view all the answers

    What is the purpose of the Rosetta ConnectChainsMover in the design process?

    <p>To loop scaffold backbones</p> Signup and view all the answers

    The resulting designs were influenced by the threaded target sequence and the _______ filtering process.

    <p>gate</p> Signup and view all the answers

    What was the primary outcome of using RFdiffusion in the study?

    <p>Creating high-affinity binders from random noise</p> Signup and view all the answers

    What percentage did the average contact molecular surface for the partially diffused GCG binders increase?

    <p>33%</p> Signup and view all the answers

    What was the limit of detection (LOD) for the lucCagePTH biosensor?

    <p>10 nM</p> Signup and view all the answers

    The designed GCG binder exhibited lower recovery rates than the monoclonal antibody when normalized to 100%.

    <p>False</p> Signup and view all the answers

    What advantage do the RFdiffusion-generated binders offer over traditional antibodies?

    <p>Greater robustness and stability</p> Signup and view all the answers

    What approach provides a general route for creating high-shape-complementary binders?

    <p>RFdiffusion de novo design</p> Signup and view all the answers

    The PTH lucCage biosensor underwent a conformational change in the presence of _____ peptide target.

    <p>PTH</p> Signup and view all the answers

    The lucCagePTH sensor is designed for immunosensor applications.

    <p>False</p> Signup and view all the answers

    The designed binders provide a more robust platform for diagnostics compared to traditional _____ sensors.

    <p>immuno</p> Signup and view all the answers

    Match the following peptide binders with their corresponding recovery rates:

    <p>PTH binder = 53% GCG binder = 91.1% Antibody beads = 100% Unrelated binder = Lower recovery</p> Signup and view all the answers

    Which property of designed binders contributes to cost reduction?

    <p>Lower production costs</p> Signup and view all the answers

    What computational method is mentioned for designing helical peptide binders?

    <p>Rosetta ddG</p> Signup and view all the answers

    Designed scaffolds are less amenable to incorporation into sensors than antibodies.

    <p>False</p> Signup and view all the answers

    During the experimentation, the trypsin digestion step was _____ for the GCG binder.

    <p>skipped</p> Signup and view all the answers

    What platform is used for detecting low-abundance protein biomarkers?

    <p>LC–MS/MS</p> Signup and view all the answers

    What were the recovery rates of PTH in buffer and human plasma?

    <p>53% in buffer and 43% in human plasma</p> Signup and view all the answers

    What does the term 'partial diffusion' refer to in the context of protein design?

    <p>A technique for enhancing structural diversity and binding affinity</p> Signup and view all the answers

    The GCG binder showed comparable binding affinities to both GCG and SCT.

    <p>False</p> Signup and view all the answers

    What is the significance of adding noise during the partial diffusion process?

    <p>It allows for diversification of the starting protein fold and improves binding affinities.</p> Signup and view all the answers

    The binding affinity of the final designs to NPY was measured to be ______ nM.

    <p>5.6</p> Signup and view all the answers

    What is a primary advantage of using Hallucination for binder design?

    <p>It enables the design of binders to peptides in different conformations.</p> Signup and view all the answers

    The Bid peptide is structured even in isolation.

    <p>False</p> Signup and view all the answers

    Match the following terms with their descriptions:

    <p>Kd = Dissociation constant reflecting binding affinity AF2 = A model used for protein structure prediction FP = Fluorescence polarization, a method to measure binding Ile13 = An isoleucine residue that enhances binding through shape complementarity</p> Signup and view all the answers

    Which of the following statements accurately describes the outcomes of applying partial diffusion?

    <p>It allows small structural modifications that enhance protein interaction.</p> Signup and view all the answers

    What was the purpose of optimizing for confident binding to the target peptide during the design process?

    <p>To ensure that the designed binders effectively bind to the Bid peptide.</p> Signup and view all the answers

    The Bid peptide showed increased __________ on interaction with Hallucinated proteins.

    <p>helicity</p> Signup and view all the answers

    The designs created from the original Inpainted binders showed poorer binding efficacy after undergoing partial diffusion.

    <p>False</p> Signup and view all the answers

    Match the following terms with their descriptions:

    <p>Hallucination = Design method optimizing for binding metrics Bid peptide = Apoptosis-related protein subject to binding designs RFdiffusion = Generative process for protein structure refinement Monte Carlo search = Method used for exploring sequence space</p> Signup and view all the answers

    What does RMSD stand for, and how was it relevant in assessing the binding models?

    <p>RMSD stands for Root Mean Square Deviation; it was used to measure the alignment accuracy of the modeled structures against crystal structures.</p> Signup and view all the answers

    What was the observed result of the binding affinity measurements?

    <p>The binder had a binding affinity of 7 nM to the Bid peptide.</p> Signup and view all the answers

    The crystal structure alignments for the GCG binder indicated a shift of _____ Å towards the target in the binder backbone.

    <p>2.7</p> Signup and view all the answers

    What percentage of the highest-affinity designs was successful in binding GCG in initial screening?

    <p>25%</p> Signup and view all the answers

    Four of the Hallucinated designs were further characterized and showed soluble, monomeric expression.

    <p>True</p> Signup and view all the answers

    What is one of the challenges associated with traditional Rosetta-based design approaches?

    <p>Deep conformational sampling is very compute intensive.</p> Signup and view all the answers

    The binding affinity of the original Inpainted GCG binder was higher than that of the partially diffused binder.

    <p>False</p> Signup and view all the answers

    What structural feature was introduced at residue 13 to improve shape complementarity in the GCG binder?

    <p>Isoleucine was introduced to replace phenylalanine.</p> Signup and view all the answers

    During the reverse diffusion process of RFdiffusion, random __________ is taken as input.

    <p>Gaussian noise</p> Signup and view all the answers

    The analysis showed a contact molecular surface increase from _____ Ų to _____ Ų due to partial diffusion.

    <p>431, 522</p> Signup and view all the answers

    What unique feature do many of the binders from this method contain?

    <p>A well-defined groove</p> Signup and view all the answers

    RFdiffusion is less compute efficient than Hallucination.

    <p>False</p> Signup and view all the answers

    Which of the following proteins did the study utilize to understand specificity changes mediated by partial diffusion?

    <p>Colicin–immunity protein system</p> Signup and view all the answers

    What methodology was used to experimentally test the Hallucinated designs?

    <p>Co-expression of a GFP-tagged Bid peptide and His-tagged binders.</p> Signup and view all the answers

    Partial diffusion can only be applied to existing crystal structures.

    <p>False</p> Signup and view all the answers

    Many designed binders were predicted to bind to Bid in a predominantly __________ conformation.

    <p>helical</p> Signup and view all the answers

    Which protein was used as a comparison for binding affinity?

    <p>Bcl-2 protein Mcl-1</p> Signup and view all the answers

    What is the primary goal of RFdiffusion in the binder design process?

    <p>To design binders completely de novo</p> Signup and view all the answers

    RFdiffusion consistently requires the topology of binding proteins to be specified.

    <p>False</p> Signup and view all the answers

    What type of secondary structure do the designed binders exhibit according to circular dichroism data?

    <p>Helical secondary structure</p> Signup and view all the answers

    RFdiffusion showed high specificity, as no binding was observed to ______.

    <p>PTHrp</p> Signup and view all the answers

    Match the binder types with their corresponding affinity measurements:

    <p>PTH binder = &lt; 500 pM Bim binder = &lt; 500 pM PYY binder = 24.5 nM GCG binder = lower affinity</p> Signup and view all the answers

    What information was initially provided to RFdiffusion for designing binders?

    <p>Sequence and structures of peptides</p> Signup and view all the answers

    RFdiffusion can generate folded structures using only the amino acid sequence as input.

    <p>True</p> Signup and view all the answers

    What feature of the designed binders indicates that they are structurally stable?

    <p>Stability at 95 °C</p> Signup and view all the answers

    The designed PTH and Bim binders were found to have a ______ affinity.

    <p>picomolar</p> Signup and view all the answers

    Match the design methodologies with their outcomes:

    <p>Fixed target structure RFdiffusion = Designed high-affinity binders Flexible backbone RFdiffusion = Lower affinity than fixed structure Deep learning methods = Converged on helical peptide-binding designs Yeast surface display = Tested binding of designed peptides</p> Signup and view all the answers

    What aspect did RFdiffusion incorporate to increase its effectiveness?

    <p>Training on multi-chain systems from PDB</p> Signup and view all the answers

    The X-ray crystal structure of the Bim binder closely matched its design model.

    <p>True</p> Signup and view all the answers

    What peptide was used to demonstrate flexibility in the binding process with RFdiffusion?

    <p>PYY</p> Signup and view all the answers

    The approach used to design binders to PYY resulted in ______ affinity than that in the fixed structure case.

    <p>lower</p> Signup and view all the answers

    Study Notes

    Peptide Hormone Binding Protein Design

    • Peptide hormones, like parathyroid hormone (PTH), neuropeptide Y (NPY), glucagon (GCG), and secretin (SCT), adopt α-helical structures upon binding receptors and are crucial biomarkers.
    • Sensitive and specific quantification of these hormones currently relies on antibodies, which can be problematic due to resource intensity, low affinity, instability, and reproducibility.
    • Antibodies are not well-suited for binding extended helical peptides; anti-peptide antibodies usually bind in non-helical conformations.

    Computational Design Approaches

    • Parametric approach: Designs helical bundle scaffolds with an open groove to fit helical peptides. This helps achieve high-affinity and specific binding, with helices limiting self-association. The method samples various supercoiling and helix-helix spacings.
    • Deep learning (Hallucination): Generates helical peptide binders without pre-specifying scaffold geometries. Optimized metrics for binding, though adversarial protein sequences can be a concern. This allows design to various conformations.
    • RFdiffusion: An extension of the denoising diffusion method that directly generates protein structures. It's more efficient. Partial diffusion allows backbone resampling and refinement. This can enhance binding affinity, enabling subtle adjustments to match target shape.

    Design Method Details

    • Parametric designs: Random sampling of helical bundle scaffold parameters (supercoiling, helix distances) creates diverse models.
    • Hallucination: Initial structure predicted from sequence, optimizing in sequence space for desired binding properties with AF2 or RoseTTAFold. Refinement using ProteinMPNN to avoid unwanted sequences.
    • RFdiffusion: Starting from random noise, iteratively refines to a new protein structure to match the target (or refined using partial noise).

    Experimental Validation

    • High-affinity binders (picomolar range): Achieved through parametric, deep learning, and RFdiffusion approaches for various peptide targets (PTH, GCG, NPY, Bim).
    • Binding assays: NanoBiT split-luciferase, fluorescence polarization (FP), yeast surface display, and LC-MS/MS.

    Biosensors and Detection Tools

    • PTH lucCage biosensor: Grafting the binder into a lucCage system creates a sensitive biosensor (10 nM limit of detection) with a ≈21-fold luminescence activation in the presence of PTH.
    • LC-MS/MS enrichment: Successfully enriched PTH and GCG from buffer and human plasma. Designed binders showed high and maintained binding activity in repeated experiments avoiding antibody limitations.

    Overall Significance

    • Computationally designed helical scaffolds surpass the typical limitations of antibodies for binding helical peptides: wider surface area, easier integration with sensors, more stability, and low cost using E. coli.

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    Description

    Explore the cutting-edge methods in designing peptide hormone binding proteins. This quiz covers the significance of peptide hormones, the limitations of traditional antibody binding, and innovative computational design approaches like parametric design and deep learning. Test your knowledge on these key concepts in biochemistry.

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