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Hydrophilicity plot
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A hydrophilicity plot is a quantitative analysis of the degree of hydrophobicity or hydrophilicity of amino acids of a protein. It is used to characterize or identify possible structure or domains of a protein.
The plot has amino acid sequence of a protein on its x-axis, and degree of hydrophobicity and hydrophilicity on its y-axis. There are a number of methods to measure the degree of interaction of polar solvents such as water with specific amino acids. For instance, the Kyte-Doolittle scale indicates hydrophobic amino acids, whereas the Hopp-Woods scale measures hydrophilic residues.
Analyzing the shape of the plot gives information about partial structure of the protein. For instance, if a stretch of about 20 amino acids shows positive for hydrophobicity, these amino acids may be part of alpha-helix spanning across a lipid bilayer, which is composed of hydrophobic fatty acids. On the converse, amino acids with high hydrophilicity indicate that these residues are in contact with solvent, or water, and that they are therefore likely to reside on the outer surface of the protein.
| Amino Acid | One Letter Code | Hydropathy Score |
|---|---|---|
| Isoleucine | I | 4.5 |
| Valine | V | 4.2 |
| Leucine | L | 3.8 |
| Phenylalanine | F | 2.8 |
| Cysteine | C | 2.5 |
| Methionine | M | 1.9 |
| Alanine | A | 1.8 |
| Glycine | G | -0.4 |
| Threonine | T | -0.7 |
| Serine | S | -0.8 |
| Tryptophan | W | -0.9 |
| Tyrosine | Y | -1.3 |
| Proline | P | -1.6 |
| Histidine | H | -3.2 |
| Glutamic acid | E | -3.5 |
| Glutamine | Q | -3.5 |
| Aspartic acid | D | -3.5 |
| Asparagine | N | -3.5 |
| Lysine | K | -3.9 |
| Arginine | R | -4.5 |
The data in the above table was generated using a computer program that evaluates the average hydrophobicity of segments within a protein and uses data collected from literature.
References
[edit]- ^ Kyte, J; Doolittle, R. F. (1982). "A simple method for displaying the hydropathic character of a protein". Journal of Molecular Biology. 157 (1): 105–32. CiteSeerX 10.1.1.458.454. doi:10.1016/0022-2836(82)90515-0. PMID 7108955.
External links
[edit]Hydrophilicity plot
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Fundamentals
Definition and Purpose
A hydrophilicity plot is a graphical representation that quantifies the hydrophilic (water-attracting) or hydrophobic (water-repelling) character of amino acids along the primary sequence of a protein.[1] This visualization assigns numerical values to each residue based on its physicochemical properties, allowing for the assessment of regional tendencies toward water interaction or avoidance within the sequence. The primary purpose of a hydrophilicity plot is to aid in the prediction of protein secondary and tertiary structures, particularly by highlighting potential surface-exposed hydrophilic regions, buried hydrophobic cores, or transmembrane helices in membrane proteins.[6] In the absence of experimental structural data, such as from X-ray crystallography or NMR, these plots provide an initial computational framework for inferring folding patterns and functional domains.[1] For instance, stretches of high hydrophilicity often indicate regions likely to be solvent-accessible, while hydrophobic segments suggest involvement in the protein interior or lipid bilayers. Hydrophilicity plots originated in the early 1980s as part of pioneering bioinformatics efforts to model protein folding computationally, without relying on direct experimental observation.[1] A foundational method was introduced by Hopp and Woods in 1981, which formalized the use of sliding window averages to smooth the plot and reveal structural motifs, particularly for identifying antigenic determinants. The basic workflow entails inputting a protein's amino acid sequence, applying a hydrophilicity scale to assign values to residues, and outputting a plot where the x-axis represents residue position and the y-axis shows average hydrophilicity scores.[1] These scales, which quantify amino acid propensities, form the basis for the plot's calculations.[6]Hydrophobicity vs. Hydrophilicity
Hydrophobicity refers to the tendency of non-polar amino acids, such as leucine and valine, to avoid contact with water and aggregate in the interiors of proteins.[7] This behavior is primarily driven by the entropic gain from releasing ordered water molecules surrounding non-polar groups, supplemented by van der Waals attractions that stabilize the clustered non-polar residues.[8] In protein folding, this hydrophobic effect minimizes the exposure of non-polar surfaces to the aqueous environment, promoting the formation of compact structures.[9] In contrast, hydrophilicity describes the affinity of polar or charged amino acids, such as serine and aspartic acid, for water molecules through hydrogen bonding or electrostatic interactions.[7] These residues are typically positioned on the exterior of proteins, facilitating interactions with the solvent and enhancing overall solubility.[10] The biophysical implications of these properties are profound: the hydrophobic effect primarily stabilizes the protein core by sequestering non-polar residues away from water, while hydrophilic residues on the surface promote solubility, enable ligand binding, and support interactions at membrane interfaces.[9][7] Common examples of hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tryptophan (Trp), and proline (Pro), whose non-polar side chains lack significant hydrogen-bonding capability and thus repel water.[7] Hydrophilic amino acids encompass asparagine (Asn), glutamine (Gln), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys), histidine (His), glutamic acid (Glu), aspartic acid (Asp), lysine (Lys), and arginine (Arg); these feature polar or charged side chains that form favorable interactions with water via hydrogen bonds or ionic solvation.[7]Amino Acid Scales
Common Hydrophilicity Scales
Hydrophilicity scales provide numerical assignments to the 20 standard amino acids based on their relative affinity for water, derived from experimental measurements such as solubility in aqueous versus organic solvents, partition coefficients between immiscible phases like octanol and water, and conformational propensities observed in protein structures where residues are either buried in the hydrophobic core or exposed to solvent.[6] These scales enable quantitative assessment of hydrophilic and hydrophobic tendencies, with positive values indicating hydrophilicity and negative values indicating hydrophobicity.[11] The Hopp-Woods scale, introduced in 1981, was specifically designed to predict antigenic determinants by highlighting regions likely to be solvent-accessible on protein surfaces. It assigns positive values to hydrophilic residues based on empirical correlations with epitope locations, ranging from +3.0 (highly hydrophilic, e.g., arginine, lysine, aspartic acid, glutamic acid) to -3.4 (highly hydrophobic, e.g., tryptophan). The complete set of values for the 20 amino acids is as follows:[2]| Amino Acid | Hydrophilicity Value |
|---|---|
| Ala | -0.5 |
| Arg | 3.0 |
| Asn | 0.2 |
| Asp | 3.0 |
| Cys | -1.0 |
| Gln | 0.2 |
| Glu | 3.0 |
| Gly | 0.0 |
| His | -0.5 |
| Ile | -1.8 |
| Leu | -1.8 |
| Lys | 3.0 |
| Met | -1.3 |
| Phe | -2.5 |
| Pro | 0.0 |
| Ser | 0.3 |
| Thr | -0.4 |
| Trp | -3.4 |
| Tyr | -2.3 |
| Val | -1.5 |
Selection and Application of Scales
The selection of a hydrophilicity scale depends primarily on the analytical goal and the type of protein under study. For epitope mapping in soluble proteins, the Hopp-Woods scale excels by prioritizing charged and polar residues to highlight surface-exposed, antigenic regions. The Parker scale is similarly useful for antigenic prediction due to its basis in experimental retention times. Soluble proteins benefit from scales accentuating aqueous interactions.[15] Key factors influencing scale choice include the method of derivation, normalization range, and integration with bioinformatics software. Experimental scales, such as Hopp-Woods based on empirical epitope data, offer biophysical grounding but may vary with conditions like pH, whereas computational scales provide consistency across contexts. Normalization ensures comparability; for instance, the Hopp-Woods scale spans approximately -3.4 to +3.0.[6] Software compatibility is crucial, as tools like ProtScale support over 50 scales, allowing seamless application without manual recalculation. In practice, scales are applied via user-friendly bioinformatics platforms. ProtScale (Expasy) requires inputting a protein sequence in FASTA format, selecting a scale from a predefined list (e.g., Hopp-Woods or Parker), and specifying a sliding window size—typically 6–7 residues for fine-grained features like epitopes—to generate the plot.[17] Similarly, EMBOSS tools such as pepwindow can compute hydrophilicity-based plots by processing sequences with adjustable window parameters, outputting graphical or tabular profiles for visualization.[18] Best practices emphasize validation across multiple scales to build consensus and mitigate biases inherent to any single method. Comparing outputs from scales like Hopp-Woods and Parker often reveals overlapping hydrophilic regions, enhancing reliability in structure prediction.[6] Where post-translational modifications such as glycosylation are present, which can introduce additional hydrophilic moieties, users should adjust interpretations or supplement with modification-aware analyses, as standard scales primarily reflect unmodified amino acids.[19]Computation
Calculating Hydrophilicity Values
The calculation of hydrophilicity values for a protein sequence involves assigning numerical scores to each amino acid based on a chosen hydrophilicity scale and then averaging these scores over a sliding window to obtain a smoothed profile value at each position. This process quantifies the local hydrophilic tendency, where higher (more positive) values indicate greater hydrophilicity. The original method for hydrophilicity plots uses direct scales like the Hopp-Woods hydrophilicity scale, though inverted hydropathy scales such as the Kyte-Doolittle index can also be applied, with S(a_j) representing the scale value for the j-th amino acid (positive for hydrophilic residues such as arginine at +3.0 and negative for hydrophobic ones like isoleucine at -1.8 on the Hopp-Woods scale).[2][1] The core formula for the hydrophilicity value H_i at position i is the average over a window of n residues centered at i: This summation incorporates the scale values S(a_j) for the amino acids a_j within the window, providing a measure that smooths out single-residue fluctuations to highlight regional hydrophilic character.[1] Window sizes n are typically odd numbers between 7 and 19 residues to ensure centering and effective smoothing of local variations; smaller windows (e.g., 7) emphasize finer details in globular proteins, while larger ones (e.g., 19) better resolve extended hydrophilic or solvent-exposed segments.[17] The choice of odd n facilitates symmetric averaging around position i, reducing bias from uneven weighting. At the sequence ends, full centering is not possible, so the window is truncated to available residues, and the first value H_i is computed for the initial partial window and assigned to the central position of that window (e.g., for n=9, the first H_5 uses residues 1–9); subsequent windows advance by one residue. Padding with neutral values (S=0) is an alternative but less common approach to maintain full window size. This handling ensures the profile covers the entire sequence without artificial extensions. For illustration, consider a 9-residue peptide sequence MGSKALVPR using the Hopp-Woods hydrophilicity scale (S values: M=-1.3, G=0.0, S=0.3, K=3.0, A=-0.5, L=-1.8, V=-1.5, P=0.0, R=3.0).[2] With n=9, the single centered value H_5 is the average of all residues: Sum = -1.3 + 0.0 + 0.3 + 3.0 -0.5 -1.8 -1.5 + 0.0 + 3.0 = 1.2. Then H_5 = 1.2 / 9 ≈ 0.13, indicating slight hydrophilicity overall. For a longer sequence, the next H_6 would average residues 2–10 (shifting the window), demonstrating the sliding process.Generating the Plot
A hydrophilicity plot is typically generated as a line graph, where the x-axis represents the residue number along the protein sequence from 1 to its total length, and the y-axis displays the hydrophilicity scores, which vary by scale but often range approximately from -3.5 to +3 (e.g., for the Hopp-Woods scale).[2] The input consists of the computed hydrophilicity values for each residue position, which are then connected sequentially to form the line profile.[17] Several software tools facilitate the generation of these plots. The web-based ProtScale tool from the ExPASy server allows users to input a protein sequence in FASTA or plain text format, select a hydrophilicity scale, and automatically compute and visualize the plot as a linear graph.[17] In Python, libraries such as BioPython provide functions to calculate hydrophilicity values using amino acid scales (e.g., via Bio.SeqUtils.ProtParam.protein_scale), which can then be plotted using Matplotlib by specifying the residue positions on the x-axis and scores on the y-axis.[20] For R users, the Peptides package computes hydrophilicity indices and supports plotting via the plotIndex function, often combined with ggplot2 for customizable line graphs.[21] Customization options enhance the plot's clarity for analysis. The window averaging already provides smoothing; additional smoothing can be applied if needed, with typical window sizes ranging from 5 to 9 to balance detail and trend visibility; for instance, ProtScale defaults to a window of 9 residues centered on each position.[17] Horizontal threshold lines can be added at fixed y-values, such as 0 or scale-specific points like ±1.0, to demarcate score boundaries visually.[17] Additionally, peaks and valleys representing high or low hydrophilicity regions can be labeled with residue numbers or sequence excerpts directly on the graph using tool-specific annotation features. Output formats support various uses, including static images in PNG or PDF for reports and interactive versions via extensions like Plotly in Python for dynamic exploration.[17] Export options in tools like ProtScale and Matplotlib also include vector formats such as SVG for high-resolution publication, alongside downloadable data tables in text or CSV for further processing.[17]Interpretation
Identifying Hydrophilic and Hydrophobic Regions
In hydrophilicity plots, regions with positive y-values, typically greater than 0, are indicative of hydrophilic segments likely to be exposed on the protein surface, such as loops or epitopes that interact with solvent or form antigenic sites. Conversely, negative y-values suggest hydrophobic regions that may be buried in the protein core or span the lipid bilayer as transmembrane domains. These thresholds vary slightly by scale—such as the Hopp-Woods scale, where peaks in averaged hydrophilicity highlight potential surface-exposed areas—but generally, values above 0 confirm hydrophilic character while those below 0 signal hydrophobicity.[22] Pattern recognition in these plots relies on identifying peaks and troughs along the sequence axis. Pronounced peaks correspond to hydrophilic, exposed areas prone to solvent interaction, whereas deep troughs denote hydrophobic, buried segments shielded from water. For transmembrane predictions, continuous spans exceeding 19 residues with sustained negative values are characteristic of alpha-helical domains embedded in membranes, as this length accommodates a full helical turn sufficient for bilayer traversal.[22] The use of a sliding window in plot generation introduces averaging effects that smooth data, potentially blurring abrupt transitions between hydrophilic and hydrophobic regions. Smaller windows (e.g., 6-7 residues) enhance resolution for detecting sharp surface features but increase noise, while larger windows (19-21 residues) better resolve broad hydrophobic spans like transmembrane helices at the cost of detail in loop regions. Adjusting the window size thus allows tailoring the plot's sensitivity to the desired structural resolution. A classic case study is the hydrophilicity plot of bacteriorhodopsin, a seven-transmembrane helix protein from Halobacterium salinarum. Using the inverted Kyte-Doolittle scale (where negative values indicate hydrophobicity), the plot reveals seven distinct troughs, each spanning approximately 20-25 residues with negative values, accurately predicting the alpha-helical transmembrane domains that form the protein's light-driven proton pump structure. Intervening peaks above 0 correspond to hydrophilic loops exposed to the aqueous environment, confirming the plot's utility in delineating membrane topology without prior structural knowledge.[23]Applications in Protein Structure Prediction
Hydrophilicity plots play a key role in predicting the transmembrane topology of membrane proteins by identifying hydrophilic regions that likely form extracellular or intracellular loops between hydrophobic transmembrane helices. In proteins such as G-protein coupled receptors, these plots highlight surface-accessible domains that influence ligand binding and signaling, complementing hydropathy analyses to refine overall topology models. For instance, peaks in hydrophilicity correspond to solvent-exposed loops, aiding in the accurate delineation of membrane insertion points and orientation.[24][3] In epitope mapping, hydrophilicity plots are essential for locating potential B-cell epitopes, as these antigenic sites are predominantly found in hydrophilic surface patches accessible to antibodies. This application supports vaccine design by prioritizing regions with high local hydrophilicity for immunogenic peptide selection, and facilitates antibody engineering by predicting binding hotspots. The seminal Hopp-Woods scale has demonstrated high predictive success for antigenic determinants in proteins like hepatitis B surface antigen and influenza hemagglutinins, with hexapeptide window averages achieving optimal accuracy in experimental validations.[22][25] Hydrophilicity plots contribute to solubility assessment by quantifying the exposure of hydrophilic residues, which correlates with a protein's ability to remain soluble in aqueous environments during expression in heterologous hosts like E. coli. Regions with sustained positive hydrophilicity values indicate better folding and reduced aggregation risk, guiding engineering efforts to enhance expression yields. This is particularly valuable in biotechnology, where plots help forecast challenges in recombinant protein production based on sequence-derived surface properties.[26][10] These plots integrate with modern protein structure prediction tools, such as AlphaFold, to validate predicted solvent-exposed regions and refine homology models by incorporating hydrophilicity as a constraint for surface feature accuracy. In real-world applications, they support genome annotation in databases like UniProt, where ProtScale-generated plots identify hydrophilic patches for functional annotation, including potential drug targets in viral and bacterial proteins up to 2025. For example, in drug discovery, such analyses have aided in targeting hydrophilic epitopes on SARS-CoV-2 spike protein for therapeutic antibody development.[27][4]Limitations and Advances
Shortcomings of Traditional Plots
Traditional hydrophilicity plots, such as those based on the Hopp-Woods scale, oversimplify protein behavior by reducing complex three-dimensional structures to a one-dimensional sequence analysis, thereby ignoring spatial contexts like beta-sheet formations or dynamic conformational changes. In beta-barrel membrane proteins, for instance, alternating hydrophobic and hydrophilic residues within strands lead to averaged values near neutrality in sliding-window calculations, rendering certain regions undetectable. Similarly, the fixed window averaging (typically 6–7 residues) obscures short hydrophilic motifs or irregular secondary structures, as the smoothing effect dilutes localized signals. This approach assumes a linear correlation between sequence hydrophilicity and solvent exposure, which fails to capture the folded protein's geometry or flexibility in intrinsically disordered regions (IDPs). Hydrophilicity scales often show poor correlation with actual solvent accessibility in folded structures, as they rely on empirical propensities rather than structural data. Variability across hydrophilicity scales introduces significant biases, resulting in inconsistent predictions for the same protein sequence. Scales like Hopp-Woods, derived from observed antigenic regions, often diverge from others like Parker et al., with separation capacities between hydrophilic and surface-exposed classes limited in analyses of peptide pools. These foundations, lacking integration of modern biophysical measurements, propagate errors in cross-scale comparisons and limit reliability for diverse protein families, particularly in predicting antigenic determinants. Traditional plots exhibit high rates of false positives and negatives, particularly in non-helical membrane proteins or IDPs, where prediction accuracy for surface-exposed regions is significantly reduced. In beta-sheet dominated outer membrane proteins, false negatives arise from the method's bias toward linear hydrophilic stretches, missing barrel structures despite their prevalence in gram-negative bacteria. For IDPs, the lack of stable cores leads to overprediction of solvent exposure, with error rates amplified in flexible regions lacking defined motifs. Quantitative assessments show limited performance against experimental data like solvent accessibility from structures, underscoring inadequacy for non-globular architectures. The method's lack of contextual integration further compounds these issues, as it disregards sequence-specific motifs, evolutionary conservation, or environmental influences like pH variations. Charged residues such as aspartate or lysine exhibit pH-dependent ionization that alters effective hydrophilicity, yet fixed scales assume neutral conditions, leading to misclassifications in acidic or basic cellular compartments. Without accounting for conserved hydrophilic patches critical for function or interactions, plots fail to distinguish functional solvent-exposed regions from artifacts, reducing their utility in evolutionary or motif-driven analyses.Modern Alternatives and Improvements
Since the 2010s, machine learning integrations have revolutionized the prediction of hydrophilic and hydrophobic regions in proteins, surpassing traditional hydrophilicity plots by leveraging neural networks trained on vast sequence and structure datasets. DeepTMHMM, introduced in 2022, employs a deep learning architecture based on protein language models to predict transmembrane topologies, including alpha-helical and beta-barrel structures, with state-of-the-art accuracy exceeding 90% in segment overlap metrics for benchmark datasets. This approach implicitly accounts for hydrophilicity through sequence embeddings, enabling more precise identification of membrane-spanning hydrophobic segments and exposed hydrophilic regions without relying on explicit scale-based sliding windows.[28] Multi-scale approaches have enhanced reliability by combining multiple hydrophilicity scales or integrating them with complementary metrics, such as charge-hydropathy (CH) plots, to better predict intrinsically disordered regions. In CH plots, normalized net charge is plotted against mean hydropathy to classify proteins as ordered or disordered, with modern refinements using optimized scales like IDP-Hydropathy achieving improved binary classification accuracy over single-scale methods.[29] Consensus methods, such as TOPCONS, aggregate predictions from diverse algorithms and scales to forecast membrane protein topology, reducing false positives in hydrophilic/hydrophobic region delineation by up to 10-15% compared to individual tools.[30] Advanced deep learning tools like AlphaFold, released in 2021, have further diminished dependence on explicit hydrophilicity plots by predicting full atomic-level 3D structures from sequences alone, implicitly incorporating hydrophobicity patterns through training on experimental structures. AlphaFold's confidence scores (pLDDT) often exceed 90 for well-predicted regions, allowing direct visualization of solvent-exposed hydrophilic surfaces without intermediate plotting. Subsequent versions, such as AlphaFold3 (2024), improve predictions of interactions and dynamics, enhancing mapping of hydrophilic interfaces. Validation via cryo-electron microscopy (cryo-EM) has complemented these predictions, providing high-resolution experimental confirmation of hydrophobic cores in membrane proteins and reducing reliance on predictive plots. Looking ahead, future developments emphasize dynamic and integrative analyses, such as molecular dynamics (MD) simulations to capture hydrophilicity fluctuations over time, and open-source models like ESMFold (2022) for ultrafast structure prediction using transformer-based language models. ESMFold achieves near-AlphaFold accuracy (median LDDT difference <0.05) on over half of tested proteins, facilitating rapid assessment of hydrophilic exposures in large-scale studies.[31] Incorporation of multi-omics data, including evolutionary couplings and post-translational modifications, promises even more holistic hydrophilicity modeling beyond static sequence-based plots.[31]References
- https://web.[expasy](/page/Expasy).org/protscale/protscale-doc.html