Polypeptide Structure Research Articles

Transcriptome-wide sites of collided ribosomes reveal principles of translational pausing.

Translation initiation is the major regulatory step defining the rate of protein production from an mRNA. Meanwhile, the impact of nonuniform ribosomal elongation rates is largely unknown. Using a modified ribosome profiling protocol based on footprints from two closely packed ribosomes (disomes), we have mapped ribosomal collisions transcriptome-wide in mouse liver. We uncover that the stacking of an elongating onto a paused ribosome occurs frequently and scales with translation rate, trapping ∼10% of translating ribosomes in the disome state. A distinct class of pause sites is indicative of deterministic pausing signals. Pause site association with specific amino acids, peptide motifs, and nascent polypeptide structure is suggestive of programmed pausing as a widespread mechanism associated with protein folding. Evolutionary conservation at disome sites indicates functional relevance of translational pausing. Collectively, our disome profiling approach allows unique insights into gene regulation occurring at the step of translation elongation.

Design of BRC analogous peptides based on the complex BRC8-RAD51 and the preliminary study on the peptide structures.

BRCA2 is an important tumor suppressor gene that plays a critical role in preserving the stability of cellular genetic information, participating in DNA repair by engaging in binding interactions with RAD51 proteins. However, the lack of structural data on BRCA2 and RAD51 makes the study of their interaction mechanism still a great challenge. We characterize the structure of the BRC8-RAD51 complex using ZDOCK protein docking software and identify the potential non-conserved active site of BRC8 via virtual alanine scanning, utilizing the obtained results to synthesize BRC8, its six analogous peptides (BRC8-1 to BRC8-6), and critical peptide fragment of RAD51 (RAD51(231-260)) by Fmoc solid-phase synthesis. The analogous peptides are found to exhibit a secondary structure significantly different from that of BRC8 by circular dichroism spectroscopy, which indicates that mutation sites determined by computer-aided simulation correspond to key amino acid residues substantially affecting polypeptide structure. On the other hand, the secondary structure of RAD51(231-260) was also considerably influenced by its interaction with BRC8 and analogs, e.g., the fraction of the α-helical structure in RAD51(231-260) increased to 23.6, 15.1, and 13.5% upon interaction with BRC8-1, BRC8-3, and BRC8-6, respectively. The results show that the properties of C-terminal amino acid residues significantly influence peptide-peptide interactions, in agreement with the results of virtual alanine scanning. Therefore, computer-aided simulation was confirmed to be a technique that is useful for narrowing down the range of sites responsible for interactions between peptides or proteins, and provides new inspirations for the design of peptides with strong interactions.

Inter‐domain Protein‐Protein Interaction through a Unique α‐Helix in the Hinge Region of E coli Topoisomerase I Leads to the Conformational Change of the Enzyme

E coli topoisomerase I (EcTopI) is a type IA topoisomerase that relaxes negatively supercoiled DNA to prevent the inhibition of vital cellular processes such as transcription. Type IA topoisomerase polypeptide folds into a torus structure to catalyze the breaking and rejoining of a DNA single‐strand coupled with DNA strand passage, thus maintain the DNA topology. Previously elucidated full‐length crystal structure of EcTopI consisting nine domains (D1–D9), bound with an ssDNA to the C‐terminal domain revealed that D6 has an unusual 21‐amino acids insert in the middle of the first β‐strand. A part of the insert (12 amino acids) forms a unique alpha helix (α1), which is present in the vicinity of the N‐terminal domain at the hinge region of D2 and D4. We hypothesized that the C‐terminal element D6 domain interacts with the N‐terminal elements D2 and D4 domains hinge region through this α1‐helix and aids in the separation of D3 from D4 and D1, thus creating an opening to the toroid hole for DNA strand passage in the catalytic cycle. Site‐directed mutagenesis has been employed to create two mutant EcTopI proteins with deletion of D6‐ α1 helix (residues 647–658, Mut‐1) and substitutions of A651G and A655G to perturb α1‐helix structure (Mut‐2). Biochemical analysis of these mutant proteins showed significantly reduced relaxation activity with negatively supercoiled DNA as substrate, and the kinetics of DNA negative supercoil removal by mutants EcTopI showed clear decrease in the rate of relaxation at different time points versus wild‐type EcTopI. To test the effect of these mutations on protein’s thermal stability, thermal shift assay has been carried out and the result suggested that the mutations affected the inter‐domain interactions without significant destabilization of the polypeptide structure as only the first melting peak is shifted to lower temperature but no shift in second peak has been observed compared to wild‐type. Our findings suggest that D6 α1‐helix supports a functional role in the hinge regions between D2 and D4 where its movement (e.g. a push or a pull) as an arm against the hinge will cause the rotation of D2 in respect to D4 leading to the enzyme undergoing a large conformational change required for catalysis. The results of this study have provided a more in‐depth mechanistic insight into the mechanism of type IA topoisomerases that is required for maintaining DNA topology.Support or Funding InformationNIH grant R 01 GM 054226E coli topoI‐Crystal StructureFigure 1Thermal shift assayFigure 2

Structure Analysis of Amyloid Aggregates at Lipid Bilayers by Supercritical Angle Raman Microscopy.

The amyloid-β peptide is correlated with Alzheimer's disease and is assumed to cause toxicity by its interaction with the neuron membrane. A custom-made microscope objective based on the supercritical angle technique was developed by our group, which allows investigation of interfacial events by performing surface-sensitive and low-invasive spectroscopy. Applied to Raman spectroscopy, this technique was used to collect information about the structure of polypeptides that interact with a supported lipid bilayer. Notably, the conformation used by amyloid-β(1-40) and amyloid-β(1-42) when interacting directly with or next to the supported lipid bilayer was characterized. We observed two distinct secondary structures, α-helix and β-sheet, which were exhibited by the peptide. These two structures were detected simultaneously. The propensity of the peptide to fold into these structures seemed dependent on both their number of amino acids and their proximity with the supported lipid bilayer. The α-helix structure was observed for amyloid-β(1-42) fragments that were closer to the lipid bilayer. Peptides that were located further away from the bilayer favored the β-sheet structure. Amyloid-β(1-40) was less prone to adopt the α-helix secondary structure.

Hirudin Ameliorates Renal Interstitial Fibrosis via Regulating TGF-β1/Smad and NF-κB Signaling in UUO Rat Model.

Purpose Hirudin, a polypeptide structure containing 65 amino acids, is a potent natural thrombin inhibitor with anticoagulant property extracted from Hirudo medicinalis. It has been reported to have anti-inflammatory and antifibrotic property. Here we explored the renoprotective effect of hirudin on unilateral ureteral obstruction (UUO) induced renal interstitial fibrosis (RIF). Methods Rats were randomly divided into five groups: sham group, UUO alone group, and three UUO + hirudin-treatment groups (10, 20, or 40 IU/kg/d, for 14 continuous days). At the end of the experiment period, animals were sacrificed. Pathologic changes in renal specimens were observed using hematoxylin and eosin (HE) staining and Masson staining. The expressions of collagen III (Col III), fibronectin (FN), α-smooth muscle actin (α-SMA), protease-activated receptor 1 (PAR-1), and proteins in the TGF-β1/Smad and NF-κB pathways in renal tissues were examined by immunohistochemistry and/or Western blotting. Results HE and Masson staining showed that hirudin-treated UUO rats had lower extent of renal injury and deposition of extracellular matrix (ECM) in renal interstitium than those in the UUO group. The results of immunohistochemistry and WB indicated decreased protein expressions of Col III, FN, α-SMA, PAR-1, and inflammatory markers such as tumor necrosis factor-α and interleukin-6 after hirudin treatment. Furthermore, hirudin reduced the expressions of transforming growth factor β1 (TGF-β1), phosphorylated-Smad2, and phosphorylated-Smad3 in the UUO model. In parallel, we found inhibited nuclear factor-κB (NF-κB) signaling after hirudin treatment, with downregulated protein expressions of P65, phosphorylated-P65, and phosphorylated-iκBα and increased iκBα. Conclusion Hirudin improves kidney injury and suppresses inflammatory response and ECM accumulation in UUO rats; its underlying mechanism may be associated with the inhibition of TGF-β1/Smad and NF-κB signaling.

Supramolecular secondary helical structures in solid-state N-protected amino acids.

Helix is an important secondary structure in proteins and polypeptides, which, however, has rarely been recognized in amino acids or their simple derivatives. In this work, we firstly unveil the generic existence of supramolecular helical secondary structures in solid-state N-protected amino acids. Throughout searching in Cambridge Structural Database followed by screening, ∼10% N-protected amino acids were evidenced to form H-bonded helical structures, thus covering 15 coded amino acids and diverse types of protecting groups. Helical structures were typically classified as 21 and 31 symmetry, and specific double-strand helical structures were discovered. Computational studies on the calculated electronic circular dichroism spectra show well-defined correlation to experimental results, indicative of the supramolecular secondary structures that possess feature Cotton effects similar to naturally occurring α-helices in proteins. Such feature Cotton effects could be transferred to protecting groups, which is of vital significance to the emerging chiroptical materials. This work highlighting the neglected structural analysis would offer a better understanding and deep insight into the structure relationship on the supramolecular chiral materials, crystal engineering and chiroptical materials based on amino acids and their derivatives.

Effect of high-intensity ultrasound treatment in combination with transglutaminase and nanoparticles on structural, mechanical, and physicochemical properties of quinoa proteins/chitosan edible films

The effect of high-intensity ultrasound (US) combined with transglutaminase treatment (TG) and the inclusion of nanoparticles (Np) on the structural, mechanical, barrier, and physicochemical properties of quinoa protein/chitosan composite edible films were evaluated. Structurally it was observed that the maximum temperatures of the thermal degradation increased with the use of combined US and TG treatment, generating films with superior thermal stability. FTIR results showed that in the amide zone I oscillations of the polypeptide structure were related to the stretching vibrations of CO in the US/TG-Np edible film. Which has generally been associated with changes in the structure and formation of covalent bonds by the action of TG. The US improved mechanical properties by increasing the tensile strength (with or without the application of TG). While combining US-TG produced a significant increase in thickness, decrease in elongation percentage, and increase in tensile strength. Which can be attributed to cross-linking produced by TG. Water vapour permeability increased in all cases. In general, the combination of US-TG treatments showed a more pronounced effect on the structure and mechanical properties.

Model of Early Stage Intermediate in Respect to Its Final Structure.

The model, describing a method of determining the structure of an early intermediate in the process of protein folding to analyze nonredundant PDB protein bases, allows determining the relationship between the sequence of tetrapeptides and their structural forms expressed by structural codes. The contingency table expressing such a relationship can be used to predict the structure of polypeptides by proposing a structural form with a precision limited to the structural code. However, by analyzing structural forms in native forms of proteins based on the fuzzy oil drop model, one can also determine the status of polypeptide chain fragments with respect to the assumptions of this model. Whether the probability distributions for both compliant and noncompliant forms were similar or whether the tetrapeptide sequences showed some differences at a level of a set of structural codes was investigated. The analysis presented here indicated that some sequences in both forms revealed differences in probability distributions expressed as a negative statistically significant correlation coefficient. This meant that the identified sections (tetrapeptides) took different forms against the fuzzy oil drop model. It may suggest that the information of the final status with respect to hydrophobic core formation is already carried by the structure of the early-stage intermediate.

Peppy: A virtual reality environment for exploring the principles of polypeptide structure

A key learning outcome for undergraduate biochemistry classes is a thorough understanding of the principles of protein structure. Traditional approaches to teaching this material, which include two-dimensional (2D) images on paper, physical molecular modeling kits, and projections of 3D structures into 2D, are unable to fully capture the dynamic 3D nature of proteins. We have built a virtual reality application, Peppy, aimed at facilitating teaching of the principles of protein secondary structure. Rather than attempt to model molecules with the same fidelity to the underlying physical chemistry as existing, research-oriented molecular modelling approaches, we took the more straightforward approach of harnessing the Unity video game physics engine. Indeed, the simplicity and limitations of our model are strengths in a teaching context, provoking questions and thus deeper understanding. Peppy allows exploration of the relative effects of hydrogen bonding (and electrostatic interactions more generally), backbone φ/ψ angles, basic chemical structure, and steric effects on a polypeptide structure in an accessible format that is novel, dynamic, and fun to use. Apart from describing the implementation and use of Peppy, we discuss the outcomes of deploying Peppy in undergraduate biochemistry courses.

Structure, Topology, and Dynamics of Membrane-Inserted Polypeptides and Lipids by Solid-State NMR Spectroscopy: Investigations of the Transmembrane Domains of the DQ Beta-1 Subunit of the MHC II Receptor and of the COP I Protein p24.

MHC class II receptors carry important function in adaptive immunity and their malfunctioning is associated with diabetes type I, chronic inflammatory diseases and other autoimmune diseases. The protein assembles from the DQ alpha-1 and DQ beta-1 subunits where the transmembrane domains of these type I membrane proteins have been shown to be involved in homo- and heterodimer formation. Furthermore, the DQ alpha 1 chain carries a sequence motif that has been first identified in the context of p24, a protein involved in the formation of COPI vesicles of the intracellular transport machinery, to specifically interact with sphingomyelin-C18 (SM-C18). Here we investigated the membrane interactions and dynamics of DQ beta-1 in liquid crystalline POPC phospholipid bilayers by oriented 15N solid-state NMR spectroscopy. The 15N resonances are indicative of a helical tilt angle of the membrane anchor sequence around 20°. Two populations can be distinguished by their differential dynamics probably corresponding the DQ beta-1 mono- and homodimer. Whereas, this equilibrium is hardly affected by the addition of 5 mole% SM-C18 a single population is visible in DMPC lipid bilayers suggesting that the lipid saturation is an important parameter. Furthermore, the DQ alpha-1, DQ beta-1 and p24 transmembrane helical domains were reconstituted into POPC or POPC/SM-C18 lipid bilayers where the fatty acyl chain of either the phosphatidylcholine or of the sphingolipid have been deuterated. Interestingly in the presence of both sphingolipid and polypeptide a strong decrease in the innermost membrane order of the POPC palmitoyl chain is observed, an effect that is strongest for DQ beta-1. In contrast, for the first time the polypeptide interactions were monitored by deuteration of the stearoyl chain of SM-C18. The resulting 2H solid-state NMR spectra show an increase in order for p24 and DQ alpha-1 which both carry the SM recognition motif. Thereby the data are suggestive that SM-C18 together with the transmembrane domains form structures imposing positive curvature strain on the surrounding POPC lipids. This effect is attenuated when SM-C18 is recognized by the protein.

Identification of New Inhibitors with Potential Antitumor Activity from Polypeptide Structures via Hierarchical Virtual Screening.

Leukemias are neoplasms that affect hematopoietic cells, which are developed by genetic alterations (mutations) that lead to the loss of proliferation control mechanisms (maturation and/or cell death). The α4β1 integrin receptor is a therapeutic target for inflammation, autoimmune diseases and lymphoid tumors. This study was carried out to search through the antagonists-based virtual screening for α4β1 receptor. Initially, seventeen (17) structures were selected (based on the inhibitory activity values, IC50) and the structure with the best value was chosen as the pivot. The pharmacophoric pattern was determined from the online PharmaGist server and resulted in a model of score value equal to 97.940 with 15 pharmacophoric characteristics that were statistically evaluated via Pearson correlations, principal component analysis (PCA) and hierarchical clustering analysis (HCA). A refined model generated four pharmacophoric hypotheses totaling 1.478 structures set of Zinc_database. After, the pharmacokinetic, toxicological and biological activity predictions were realized comparing with pivot structure that resulted in five (ZINC72088291, ZINC68842860, ZINC14365931, ZINC09588345 and ZINC91247798) structures with optimal in silico predictions. Therefore, future studies are needed to confirm antitumor potential activity of molecules selected this work with in vitro and in vivo assays.

Energy propagation along polypeptide α-helix: Experimental data and ab initio zone structure

We report new theoretical and experimental results that may significantly change our ideas on the workings of life. We performed ab initio analysis of the band structure of periodic α-helix polypeptides (PP) in function of the chain length. Three different calculation approaches were tested: (a) PP described semiempirically as a one-dimensional object, with the amino acids substituted by effective atoms; (b) density functional theory (DFT) as implemented in WIEN2k approach, and (c) CRYSTAL-17 software package. The approach (c) was used for the detailed ab initio analysis, as it provided better accuracy in less computation time. We found that the bandgap was weakly dependent on the PP composition, with the asymptotic values in the 0.43 – 0.63 eV range. We estimated the effective electron and hole masses, their mean free path and mobility for the glycine-PP. The electron mobility in the PP conductive band was about half of that in polycrystalline silicon. The PP zone structure was used to study the mechanism of energy transfer along the PP. The current-voltage (I/V) characteristics of Müller cell (MC) intermediate filaments (IFs) from porcine retina were experimentally measured. The measured I/V characteristics show that the IFs behave as semiconductors. These results were discussed in light of the presently reported PP zone structure theory. The results obtained may open new areas in biomedical research and applications.

Effect of polypeptide block length on nano-assembly morphology and thermo-sensitivity of methyl poly (ethylene glycol)-poly (L-valine) copolymer aqueous solutions

The delicacy of nature prompted scientists to create functional or responsive biomaterials using existing natural components. Here we reported novel thermo-gelling hydrogels based on methyl poly (ethylene glycol)-poly (L-valine) (mPEG-L-PV) diblock copolymer solutions, which exhibited well-defined sol–gel transition at a temperature range from 10 °C to 40 °C. The β-sheet was the dominant secondary structure for poly(L-PV) block when the molecular weight of the polypeptide ranged from 277 Da to 1138 Da. The formation of mPEG–L-PV clear sol solutions was related to the stable nanostructures that were aggregated clusters of poly(L-PV) β-sheet filaments and stabilized by the hydrophilic PEG segments. The block length of poly(L-PV) significantly affected the secondary structures of polypeptide and the morphology of assembled filaments, which resulted in different thermo-gelling behavior of the copolymer solutions. The occurrence of sol–gel transition with temperature was primarily due to the dehydration effect of PEG block, whereas in the case of the copolymer with a lower molecular weight of poly(L-PV) block, the change of polypeptide secondary structures also contributed to the formation of the gel. This paper provides fundamental information about the strong dependence of polypeptide block length on the nano-assembly morphology as well as thermosensitive behavior of polypeptide-based hydrogels.

Parallelism of stable traces

A parallel $d$-stable trace is a closed walk which traverses every edge of a graph exactly twice in the same direction and for every vertex $v$, there is no subset $X \subseteq N(v)$ with $1 \leq |N| \leq d$ such that every time the walk enters $v$ from $X$, it also exits to a vertex in $X$. In the past, $d$-stable traces were investigated as a mathematical model for an innovative biotechnological procedure -- self-assembling of polypeptide structures. Among other, it was proven that graphs that admit parallel $d$-stable traces are precisely Eulerian graphs with minimum degree strictly larger than $d$. In the present paper we give an alternative, purely combinatorial proof of this result.

Periodic introduction of aromatic units in polypeptides via chemoenzymatic polymerization to yield specific secondary structures with high thermal stability

Polypeptides containing periodic aromatic residues in their main chains were synthesized via papain-catalyzed chemoenzymatic polymerization of tripeptide ester monomers under moderate conditions in aqueous buffers. As the monomer, 4-aminobenzoic acid (Abz) was modified with Gly or Ala at the N- and C-termini to mitigate the poor substrate specificity of papain to unnatural amino acids. The tripeptide esters, namely, GlyAbzGly and AlaAbzAla ethyl esters, can be recognized and polymerized by papain, resulting in the polypeptides poly(GlyAbzGly) and poly(AlaAbzAla), respectively, with periodic sequences containing Abz units every three residues. Copolymerization of tripeptide esters with Gly or Ala ethyl ester also proceeded in the presence of papain. The secondary structures of the Abz-containing polypeptides were investigated by IR and wide-angle X-ray diffraction (WAXD) analysis. The WAXD profile of poly(GlyAbzGly) was similar to that of polyGly, whereas poly(AlaAbzAla) adopted a sheet-like structure similar to the β-sheet of polyAla. Thermal analysis of Abz-containing polypeptides revealed that the high thermal stability of the Abz-containing polypeptides is related to the distinct sequences that periodically include Abz residues.

Colloquium: Proteins: The physics of amorphous evolving matter

Thought of as inert matter, proteins present a challenge to the methods of statistical mechanics and materials science. Unlike systems in or near equilibrium, protein structure has evolved over the eons. In this Colloquium, a framework to study polypeptide structure and evolution is proposed that employs the tools of correlation and response to these adapted and highly functional biomolecules.

PEP-FOLD design, synthesis, and characteristics of finger-like polypeptides.

Polypeptides with finger-like structures can often intercalate into the grooves of DNA, thereby affecting DNA repair or activating gene transcription, both of which are crucial for the regulation of physiological processes. Their conserved amino acid sequence and simple structure have provided useful elements for the design and assembly of functional molecules. In this paper, using the C2H2 zinc finger domain and the PEP-FOLD3 online simulation platform 11 polypeptides containing 22 amino acid residues were designed. In addition, the CD spectroscopy was combined with the fluorescence spectroscopy to study the polypeptide structures and their interaction with DNA. Results showed that although addition of zinc ions affected the polypeptide structure, particularly of the polypeptides A4, B1, and B3, zinc ion was not an essential factor for increasing polypeptide-DNA interactions. Our study revealed an increase in the interaction strength between mutated polypeptides and DNA, suggesting that mutations disrupt polypeptide structure, and polypeptides interact with DNA by groove and electrostatic binding. Mutations at the 12th and 15th amino acid residues had the greatest effect. The stronger binding between A2 or B2 and DNA indicates that the polypeptide has a spatial structure that can stably interact with DNA. The structure and characteristics of these polypeptide domains can provide information for the design and development of new polypeptide functional molecules, which could have potential significance and applications. However, this information also suggests that there are many challenges facing polypeptide design due to the synergistic effects between the side chains of amino acid residues.

Synthesis of AIE polyethylene glycol-block-polypeptide bioconjugates and cell uptake assessments of their self-assembled nanoparticles

A synthetic polypeptide-based bioconjugate bearing tetraphenylethylene (TPE) at the polypeptide side chains, named mPEG-PPLGgTPE, was synthesized by ring-opening polymerization (ROP) of α-amino acid-N-carboxyanhydride (NCA) along with post-polymerization modification method. Interestingly, the bioconjugate enabled both the aggregation induced emission (AIE) effect at 469 nm and the aggregation caused quenching (ACQ) effect around 365 nm depending on water fraction in the tetrahydrofuran/water (THF/H2O) mixed solution, and this feature was very different from the commonly used TPE-based AIE polymers. The strong emission at the 365 nm was a rarely seen monomeric emission for the TPE units. Further studies on the fluorescence spectra indicated that the monomeric emission was not related with the secondary structure of polypeptides, but caused by the TPE unit itself. The mPEG-PPLGgTPE could self-assemble to AIE nanoparticles (NPs) in water in a mean size of 253 nm with polyethylene glycol (PEG) as the shells in surface. In addition, HeLa cell uptake level of the NPs was unsatisfactory according to the cell imaging results, which was independent of the polypeptides conformation, the PEG length and the size of NPs.

Mapping the amide-I vibrations of model dipeptides with secondary structure sensitivity and amino acid residue specificity, and its application to amyloid β-peptide in aqueous solution.

Vibrational spectroscopy has been known as particularly well-suited for deciphering the polypeptide's structure. To decode structural information encoded in IR spectra, we developed amide-I frequency maps on the basis of model dipeptides to correlate the amide-I frequency of interest to the combination of the calculated secondary structure dependent amide-I frequency by using DFT method and the electrostatic potentials that projected onto the amide unit from the micro-environment within molecular mechanics force field. The constructed maps were applied to model dipeptides and amyloid β-peptide fragment (Aβ25-35). The dipeptide specified map (DS map) and the hybrid map (HYB map) predicted amide-I bands of Aβ25-35 in solution satisfactorily reproduce experimental observation, and indicate the preference of forming β-sheet and random coil structure for Aβ25-35 in D2O just as the results of cluster analysis suggested. These maps with secondary structural sensitivity and amino acid residue specificity open up a way for the interpretation of amide-I vibrations and show their potentials in the understanding of molecular structure of polypeptides in solution.

Tyrosinase (TYR) gene sequencing and literature review reveals recurrent mutations and multiple population founder gene mutations as causative of oculocutaneous albinism (OCA) in Pakistani families.

To investigate eight previously unreported Pakistani families with genetically undefined OCA for mutations in TYR. Sanger sequencing of TYR has been performed in eight families with OCA phenotype. Mutation analysis was performed to establish the pathogenic role of novel mutation. Bioinformatics analysis was performed to predict the structural and functional impacts on protein due to the mutation. In this study, we identified six likely pathogenic variants of TYR (c.272 G>A, c.308 G>A, c.346C>T, c.715 C>T, c.832 C>T and c.1255 G>A), including one novel variant (c.308 G>A; p.Cys103Tyr), segregating as appropriate in each family. Cys103 lies in the highly conserved region of the tyrosinase enzyme, and p.Cys103Tyr is predicted to disturb enzymatic function via alteration of the configurational orientation of TYR leading to a more rigid polypeptide structure. We have also reviewed the mutation spectrum of TYR in Pakistani ethnicity. Published data on OCA families proposed that ~40% have been associated with genetic variations in the TYR gene. The mutations reported in this study have now been described with varying frequencies in Pakistani families, including very rare/unique mutations. A literature review of TYR gene mutations in Pakistani populations, combined with our genetic data, identified a number of gene mutations likely to represent regional ancestral founder mutations of relevance to Pakistani populations, in addition to sporadic and recurrent 'hotspot' mutations present repeatedly in other regions worldwide.