Amino Acid Sequence Modifications Research Articles

Melanocortin 1 Receptor (MC1R): Pharmacological and Therapeutic Aspects.

Melanocortins play crucial roles in regulating the stress response, inflammation, and skin pigmentation. In this review, we focus on the melanocortin 1 receptor (MC1R), a G protein-coupled receptor primarily known for regulating skin pigmentation and exhibiting anti-inflammatory effects. First, we provide an overview of the structure, signaling pathways, and related diseases of MC1R. Next, we discuss the potential therapeutic use of synthetic peptides and small molecule modulators of MC1R, highlighting the development of various drugs that enhance stability through amino acid sequence modifications and small molecule drugs to overcome limitations associated with peptide characteristics. Notably, MC1R-targeted drugs have applications beyond skin pigmentation-related diseases, which predominantly affect MC1R in melanocytes. These drugs can also be useful in treating inflammatory diseases with MC1R expression present in various cells. Our review underscores the potential of MC1R-targeted drugs to treat a wide range of diseases and encourages further research in this area.

Fluid protein fold space and its implications.

Fold-switching proteins, which remodel their secondary and tertiary structures in response to cellular stimuli, suggest a new view of protein fold space. For decades, experimental evidence has indicated that protein fold space is discrete: dissimilar folds are encoded by dissimilar amino acid sequences. Challenging this assumption, fold-switching proteins interconnect discrete groups of dissimilar protein folds, making protein fold space fluid. Three recent observations support the concept of fluid fold space: (1) some amino acid sequences interconvert between folds with distinct secondary structures, (2) some naturally occurring sequences have switched folds by stepwise mutation, and (3) fold switching is evolutionarily selected and likely confers advantage. These observations indicate that minor amino acid sequence modifications can transform protein structure and function. Consequently, proteomic structural and functional diversity may be expanded by alternative splicing, small nucleotide polymorphisms, post-translational modifications, and modified translation rates.

Pharmacokinetics of Biopharmaceuticals: Their Critical Role in Molecular Design.

Biopharmaceuticals have developed rapidly in recent years due to the remarkable progress in gene recombination and cell culture technologies. Since the basic structure of biopharmaceuticals can be designed and modified, it is possible to control the duration of action and target specific tissues and cells by kinetic modification. Amino acid sequence modifications, albumin fusion proteins, polyethylene glycol (PEG) modifications, and fatty acid modifications have been utilized to modify the duration of action control and targeting. This review first describes the position of biopharmaceuticals, and then the kinetics (absorption, distribution, metabolism, elimination, and pharmacokinetics) of classical biopharmaceuticals and methods of drug quantification. The kinetic innovations of biopharmaceuticals are outlined, including insulin analog, antibody-related drugs (monoclonal antibodies, Fab analogs, Fc analogs, Fab-PEG conjugated proteins, antibody-drug conjugates, etc.), blood coagulation factors, interferons, and other related drugs. We hope that this review will be of use to many researchers interested in pharmaceuticals derived from biological components, and that it aids in their knowledge of the latest developments in this field.

A novel family of non-secreted tridecaptin lipopeptide produced by Paenibacillus elgii.

Bacteria from the genus Paenibacillus make a variety of antimicrobial compounds, including lipopeptides produced by a non-ribosomal synthesis mechanism (NRPS). In the present study, we show the genomic and phenotypical characterization of Paenibacillus elgii AC13 which makes three groups of small molecules: the antimicrobial pelgipeptins and two other families of peptides that have not been described in P. elgii. A family of lipopeptides with [M + H]+ 1664, 1678, 1702, and 1717m/z was purified from the culture cell fraction. Partial characterization revealed that they are similar to tridecaptin from P. terrae. However, they present amino acid chain modifications in positions 3, 7, and 10. These new variants were named tridecaptin G1, G2, G3, and G4. Furthermore, a gene cluster was identified in P. elgii AC13 genome, revealing high similarity to the tridecaptin-NRPS gene cluster from P. terrae. Tridecaptin G1 and G2 showed in vitro antimicrobial activity against Escherichia coli, Klebsiella pneumonia (including a multidrug-resistant strain), Staphylococcus aureus, and Candida albicans. Tri G3 did not show antimicrobial activity against S. aureus and C. albicans at all tested concentrations. An intriguing feature of this family of lipopeptides is that it was only observed in the cell fraction of the P. elgii AC13 culture, which could be a result of the amino acid sequence modifications presented in these variants.

Microbial characterisation and Cold-Adapted Predicted Protein (CAPP) database construction from the active layer of Greenland's permafrost.

ABSTRACTPermafrost represents a reservoir for the biodiscovery of cold-adapted proteins which are advantageous in industrial and medical settings. Comparisons between different thermo-adapted proteins can give important information for cold-adaptation bioengineering. We collected permafrost active layer samples from 34 points along a proglacial transect in southwest Greenland. We obtained a deep read coverage assembly (>164x) from nanopore and Illumina sequences for the purposes of i) analysing metagenomic and metatranscriptomic trends of the microbial community of this area, and ii) creating the Cold-Adapted Predicted Protein (CAPP) database. The community showed a similar taxonomic composition in all samples along the transect, with a solid permafrost-shaped community, rather than microbial trends typical of proglacial systems. We retrieved 69 high- and medium-quality metagenome-assembled clusters, 213 complete biosynthetic gene clusters and more than three million predicted proteins. The latter constitute the CAPP database that can provide cold-adapted protein sequence information for protein- and taxon-focused amino acid sequence modifications for the future bioengineering of cold-adapted enzymes. As an example, we focused on the enzyme polyphenol oxidase, and demonstrated how sequence variation information could inform its protein engineering.

Fluorescent proteins of the EosFP clade: intriguing marker tools with multiple photoactivation modes for advanced microscopy.

Optical fluorescence microscopy has taken center stage in the exploration of biological structure and dynamics, especially on live specimens, and super-resolution imaging methods continue to deliver exciting new insights into the molecular foundations of life. Progress in the field, however, crucially hinges on advances in fluorescent marker technology. Among these, fluorescent proteins (FPs) of the GFP family are advantageous because they are genetically encodable, so that live cells, tissues or organisms can produce these markers all by themselves. A subclass of them, photoactivatable FPs, allow for control of their fluorescence emission by light irradiation, enabling pulse-chase imaging and super-resolution microscopy. In this review, we discuss FP variants of the EosFP clade that have been optimized by amino acid sequence modification to serve as markers for various imaging techniques. In general, two different modes of photoactivation are found, reversible photoswitching between a fluorescent and a nonfluorescent state and irreversible green-to red photoconversion. First, we describe their basic structural and optical properties. We then summarize recent research aimed at elucidating the photochemical processes underlying photoactivation. Finally, we briefly introduce various advanced imaging methods facilitated by specific EosFP variants, and show some exciting sample applications.

Immunogenicity Risk Assessment for an Engineered Human Cytokine Analogue Expressed in Different Cell Substrates.

The purpose of this article is to illustrate how performance of an immunogenicity risk assessment at the earliest stage of product development can be instructive for critical early decision-making such as choice of host system for expression of a recombinant therapeutic protein and determining the extent of analytical characterization and control of heterogeneity in co- and post-translational modifications. Application of a risk-based approach for a hypothetical recombinant DNA analogue of a human endogenous cytokine with immunomodulatory functions is described. The manner in which both intrinsic and extrinsic factors could interact to influence the relative scale of risk associated with expression in alternative hosts, namely Chinese hamster ovary (CHO) cells, Pichia pastoris, Escherichia coli, or Nicotinia tabacum is considered in relation to the development of the investigational product to treat an autoimmune condition. The article discusses how particular product-related variants (primary amino acid sequence modifications and post-translational glycosylation or other modifications) and process-derived impurities (host cell proteins, endotoxins, beta-glucans) associated with the different expression systems might influence the impact of immunogenicity on overall clinical benefit versus risk for a therapeutic protein candidate that has intrinsic MHC Class II binding potential. The implications of the choice of expression system for relative risk are discussed in relation to specific actions for evaluation and measures for risk mitigation, including use of in silico and in vitro methods to understand intrinsic immunogenic potential relative to incremental risk associated with non-human glycan and protein impurities. Finally, practical guidance on presentation of this information in regulatory submissions to support clinical development is provided.

Structural modifications of amino acid sequences of radiolabeled peptides for targeted tumor imaging

Molecular imaging techniques are increasingly being used in localization, staging and therapy control of cancer. Due to their unique target specificity for the endogenous receptors, radiopeptides have been used widely for the development of radiopharmaceuticals for targeted tumor imaging in nuclear oncology. It is necessary to modify radiolabeled peptides in order to achieve more effective agents. Structural modifications of amino acid chains have significant effect on the metabolic stability, biological activity and efficiency of peptide conjugates that are currently applied as imaging tracers. There are several ways to modify the peptide chain but the most common strategies include amino acid substitutions, cyclization and multimerization. In this review, we have focused on studies involving these kind of modifications on amino acid sequences of radiolabeled peptides and we have provided an overview of the effects of these chemical modifications on the in vitro and in vivo properties of these radioconjugates and their potential as SPECT (Single photon emission computed tomography) and PET (positron emission tomography) imaging agents.

Molecular Modification of Fluoroquinolone-Biodegrading Enzymes Based on Molecular Docking and Homology Modelling.

To improve the biodegradation efficiency of fluoroquinolone antibiotics during sewage treatment, fluoroquinolone aerobic, anaerobic and facultative degrading enzymes for fluoroquinolone degradation were modified by molecular docking and homology modelling. First, amino acid residues of the binding sites of degrading enzymes for the target fluoroquinolones ciprofloxacin (CIP), norfloxacin (NOR) and ofloxacin (OFL) were analysed by the molecular docking method. The hydrophobic amino acid residues within 5 Å of the target fluoroquinolone molecules were selected as the modification sites. The hydrophobic amino acid residues at the modified sites were replaced by the hydrophilic amino acid residues, and 150 amino acid sequence modification schemes of the degrading enzymes were designed. Subsequently, a reconstruction scheme of the degrading enzyme amino acid sequence reconstruction scheme was submitted to the SWISS-MODEL server and a selected homology modelling method was used to build a new structure of the degrading enzyme. At the same time, the binding affinities between the novel degrading enzymes and the target fluoroquinolones (represented by the docking scoring function) were evaluated by the molecular docking method. It was found that the novel enzymes can simultaneously improve the binding affinities for the three target fluoroquinolones, and the degradation ability of the six modification schemes was increased by more than 50% at the same time. Among the novel enzymes, the affinity effect of the novel anaerobic enzyme (6-1) with CIP, NOR and OFL was significantly increased, with increases of 129.24%, 165.06% and 169.59%, respectively, followed by the facultative enzyme and aerobic enzyme. In addition, the designed degrading enzymes had certain selectivity for the degradation of the target quinolone. Among the novel enzymes, the binding affinities of the novel anaerobic enzyme (6-3) and CIP, the novel aerobic enzyme (3-6) and NOR, and the novel facultative enzyme (13-6) and OFL were increased by 149.71%, 178.57% and 297.12% respectively. Calculations using the Gaussian09 software revealed that the degradation reaction barrier of the novel degrading enzyme (7-1) and CIP NOR and OFL decreased by 37.65 kcal·mol−1, 6.28 kcal·mol−1 and 6.28 kcal·mol−1, respectively, which would result in efficient degradation of the target fluoroquinolone molecules. By analysing the binding affinity of the degrading enzymes before and after the modification with methanol, it was further speculated that the degradation effect of the modified aerobic degrading enzymes on organic matter was lower than that before the modification, and the increase or decrease in the degradation effect was less than 10%. The mechanism analysis found that the interaction between the modified amino acid residues of the degrading enzymes and the fluoroquinolone molecules increased. The average distance between the amino acid residues and the fluoroquinolone molecules represented a comprehensive affinity effect, and its value was positively correlated with the degradation effect of the novel degrading enzymes.

Transgenic substitution with Greater Amberjack Seriola dumerili fish insulin 2 in NOD mice reduces beta cell immunogenicity

Type I diabetes (T1D) is caused by immune-mediated destruction of pancreatic beta cells. This process is triggered, in part, by specific (aa 9–23) epitopes of the insulin Β chain. Previously, fish insulins were used clinically in patients allergic to bovine or porcine insulin. Fish and human insulin differ by two amino acids in the critical immunogenic region (aa 9–23) of the B chain. We hypothesized that β cells synthesizing fish insulin would be less immunogenic in a mouse model of T1D. Transgenic NOD mice in which Greater Amberjack fish (Seriola dumerili) insulin was substituted for the insulin 2 gene were generated (mouse Ins1−/− mouse Ins2−/− fish Ins2+/+). In these mice, pancreatic islets remained free of autoimmune attack. To determine whether such reduction in immunogenicity is sufficient to protect β cells from autoimmunity upon transplantation, we transplanted fish Ins2 transgenic (expressing solely Seriola dumerili Ins2), NOD, or B16:A-dKO islets under the kidney capsules of 5 weeks old female NOD wildtype mice. The B:Y16A Β chain substitution has been previously shown to be protective of T1D in NOD mice. NOD mice receiving Seriola dumerili transgenic islet transplants showed a significant (p = 0.004) prolongation of their euglycemic period (by 6 weeks; up to 18 weeks of age) compared to un-manipulated female NOD (diabetes onset at 12 weeks of age) and those receiving B16:A-dKO islet transplants (diabetes onset at 12 weeks of age). These data support the concept that specific amino acid sequence modifications can reduce insulin immunogenicity. Additionally, our study shows that alteration of a single epitope is not sufficient to halt an ongoing autoimmune response. Which, and how many, T cell epitopes are required and suffice to perpetuate autoimmunity is currently unknown. Such studies may be useful to achieve host tolerance to β cells by inactivating key immunogenic epitopes of stem cell-derived β cells intended for transplantation.

In vitro human helper T-cell assay to screen antibody drug candidates for immunogenicity

Monoclonal antibody (mAb) drugs offer a number of valuable treatments. Many newly developed mAb drugs include artificial modification of amino acid sequences from human origin, which may cause higher immunogenicity to induce anti-drug antibodies (ADA). If the immunogenicity of a new candidate can be understood in the nonclinical phase, clinical studies will be safer and the success rate of development improved. Empirically, in vitro immunogenicity assays with human cells have proved to be sufficiently sensitive to nonhuman proteins, but not to human/humanized mAb. To detect the weaker immunogenicity of human-based mAb, a more sensitive biomarker for in vitro assays is needed. The in vitro study here developed a proliferation assay (TH cell assay) using flow cytometry analysis that can detect a slight increase in proliferating TH cells. Samples from 218 donors treated with a low-immunogenic drug (etanercept) were measured to determine a positive threshold level. With this threshold, positive donor percentages among PBMC after treatment with higher-immunogenicity mAb drugs were noted, that is, 39.5% with humanized anti-human A33 antibody (hA33), 27.3% with abciximab, 25.9% with adalimumab, and 14.8% with infliximab. Biotherapeutics with low immunogenicity yielded values of 0% for basiliximab and 3.7% for etanercept. These data showed a good comparability with previously reported incidences of clinical ADA with the evaluated drugs. Calculations based on the data here showed that a TH cell assay with 40 donors could provide statistically significant differences when comparing low- (etanercept) versus highly immunogenic mAb (except for infliximab). Based on the outcomes here, for screening purposes, a practical cutoff point of 3/20 positives with 20 donors was proposed to alert immunogenicity of mAb drug candidates.

Improving insect control protein activity for GM crops: A case study demonstrating that increased target insect potency can be achieved without impacting mammalian safety

Many insect-protected crops express insecticidal crystal (Cry) proteins derived from the soil bacterium Bacillus thuringiensis (Bt), including both naturally-occurring Cry proteins and chimeric Cry proteins created through biotechnology. The Cry51Aa2 protein is a naturally-occurring Cry protein that was modified to increase its potency and expand its insect activity spectrum through amino acid sequence changes. The improved Cry51Aa2 variant, Cry51Aa2.834_16, and other developmental variants belong to the ETX_MTX2 family of proteins but share a low level of sequence similarity to other members of this family. This similarity is largely localized to the pore-forming and oligomerization protein domains, while sequence divergence is observed within the head domain that confers receptor binding specificity. The intact Cry51Aa2.834_16 protein was heat labile at temperatures ≥55 °C, and was rapidly degraded after exposure to the gastrointestinal protease pepsin. No acute oral toxicity was observed in mice for three protein variants of Cry51Aa2, including Cry51Aa2.834_16, at doses exceeding 1000 mg/kg body weight. The weight-of-evidence therefore supports the conclusion of safety for Cry51Aa2.834_16 and demonstrates that amino acid sequence modifications can be used to substantially increase insecticidal activity of a protein without an increased hazard to mammals.

In Vivo Characterization of the Activation and Interaction of the VanR-VanS Two-Component Regulatory System Controlling Glycopeptide Antibiotic Resistance in Two Related Streptomyces Species.

The VanR-VanS two-component system is responsible for inducing resistance to glycopeptide antibiotics in various bacteria. We have performed a comparative study of the VanR-VanS systems from two streptomyces strains, Streptomyces coelicolor and Streptomyces toyocaensis, to characterize how the two proteins cooperate to signal the presence of antibiotics and to define the functional nature of each protein in each strain background. The results indicate that the glycopeptide antibiotic inducer specificity is determined solely by the differences between the amino acid sequences of the VanR-VanS two-component systems present in each strain rather than by any inherent differences in general cell properties, including cell wall structure and biosynthesis. VanR of S. coelicolor (VanRsc) functioned with either sensor kinase partner, while VanR of S. toyocaensis (VanRst) functioned only with its cognate partner, S. toyocaensis VanS (VanSst). In contrast to VanRsc, which is known to be capable of phosphorylation by acetylphosphate, VanRst could not be activated in vivo independently of a VanS sensor kinase. A series of amino acid sequence modifications changing residues in the N-terminal receiver (REC) domain of VanRst to the corresponding residues present in VanRsc failed to create a protein capable of being activated by VanS of S. coelicolor (VanSsc), which suggests that interaction of the response regulator with its cognate sensor kinase may require a region more extended than the REC domain. A T69S amino acid substitution in the REC domain of VanRst produced a strain exhibiting weak constitutive resistance, indicating that this particular amino acid may play a key role for VanS-independent phosphorylation in the response regulator protein.

Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum

Two NADPH-cytochrome P450 reductase (CPR) genes (CaCPR1 and CaCPR2) were isolated from hot pepper (Capsicum annuum L. cv. Bukang). At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. It gradually increased during fruit ripening. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues. To investigate the enzymatic properties of CaCPR1, the cDNA of CaCPR1 was heterologously expressed in Escherichia coli without any modification of amino acid sequences, and CaCPR1 was purified. The enzymatic properties of CaCPR1 were confirmed using cytochrome c and cytochrome b5 as protein substrates. The CaCPR1 could support human CYP1A2-catalyzed reaction. It also reduced tetrazolium salts and ferricyanide. These results show that CaCPR1 is the major CPR in most hot pepper tissues. It is suggested that the CaCPR1 can be used a prototype for studying biological functions and biotechnological applications of plant CPRs.

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.

Evolutionary Reconstructions of the Transferrin Receptor of Caniforms Supports Canine Parvovirus Being a Re-emerged and Not a Novel Pathogen in Dogs

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host.

Emergence of scale-free distribution in protein–protein interaction networks based on random selection of interacting domain pairs

Recent analyses of biological and artificial networks have revealed a common network architecture, called scale-free topology. The origin of the scale-free topology has been explained by using growth and preferential attachment mechanisms. In a cell, proteins are the most important carriers of function, and are composed of domains as elemental units responsible for the physical interaction between protein pairs. Here, we propose a model for protein–protein interaction networks that reveals the emergence of two possible topologies. We show that depending on the number of randomly selected interacting domain pairs, the connectivity distribution follows either a scale-free distribution, even in the absence of the preferential attachment, or a normal distribution. This new approach only requires an evolutionary model of proteins (nodes) but not for the interactions (edges). The edges are added by means of random interaction of domain pairs. As a result, this model offers a new mechanistic explanation for understanding complex networks with a direct biological interpretation because only protein structures and their functions evolved through genetic modifications of amino acid sequences. These findings are supported by numerical simulations as well as experimental data.

Development of Prolactin Receptor Antagonists: Same Goal, Different Ways

Hyperprolactinemia is an endocrine pathology resulting from over-production of prolactin (PRL) by pituitary adenomas, and leading to various reproductive disorders. In addition, there is increasing evidence that PRL acts as a growth-promoter of breast and prostate tumors. Classical drugs blocking pituitary PRL production are not necessarily efficient in these pathological situations, which has encouraged the search for alternative ways of inhibiting the undesirable actions of PRL. Prolactin receptor (PRLR) antagonists, acting at the level of receptor activation rather than PRL production, are the most promising strategy. Based on the protein core of human (h)PRL or growth hormone (hGH), the other natural hPRLR ligand, a series of new variants have been engineered within the past couple of years, leading to various patent applications. Modifications of amino acid sequences involve single/multiple substitutions, truncations, or generation of fusion proteins. Three mechanisms of action have been reported for these PRLR antagonists: 1) inhibition of PRLR signaling by competition with endogenous PRL for receptor binding, 2) activation of specific PRLR signalling pathways resulting in actions opposite to those of wild-type hPRL, and 3) engineering of chimeric ligands targeting more than one receptor/cell type, in order to improve tumor-growth inhibition. Since none of these patented molecules is yet in clinical trials, their efficacy to treat PRL-dependent pathologies remains to be demonstrated in humans. Keywords: Breast cancer, prostate cancer, hyperprolactinemia, growth hormone, G129R-hPRL, Δ1-9-G129R-hPRL, G129RhPRL, S179D-hPRL, induced-fit

Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and μLC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by μLC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip C18 and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using μLC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. μLC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by μLC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for ∼70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

Heterotachy and functional shift in protein evolution.

Study of structure/function relationships constitutes an important field of research, especially for modification of protein function and drug design. However, the fact that rational design (i.e. the modification of amino acid sequences by means of directed mutagenesis, based on knowledge of the three-dimensional structure) appears to be much less efficient than irrational design (i.e. random mutagenesis followed by in vitro selection) clearly indicates that we understand little about the relationships between primary sequence, three-dimensional structure and function. The use of evolutionary approaches and concepts will bring insights to this difficult question. The increasing availability of multigene family sequences that has resulted from genome projects has inspired the creation of novel in silico evolutionary methods to predict details of protein function in duplicated (paralogous) proteins. The underlying principle of all such approaches is to compare the evolutionary properties of homologous sequence positions in paralogs. It has been proposed that the positions that show switches in substitution rate over time--i.e., 'heterotachous sites'--are good indicators of functional divergence. However, it appears that heterotachy is a much more general process, since most variable sites of homologous proteins with no evidence of functional shift are heterotachous. Similarly, it appears that switches in substitution rate are as frequent when paralogous sequences are compared as when orthologous sequences are compared. Heterotachy, instead of being indicative of functional shift, may more generally reflect a less specific process related to the many intra- and inter-molecular interactions compatible with a range of more or less equally viable protein conformations. These interactions will lead to different constraints on the nature of the primary sequences, consistently with theories suggesting the non-independence of substitutions in proteins. However, a specific type of amino acid variation might constitute a good indicator of functional divergence: substitutions occurring at positions that are generally slowly evolving. Such substitutions at constrained sites are indeed much more frequent soon after gene duplication. The identification and analysis of these sites by complementing structural information with evolutionary data may represent a promising direction to future studies dealing with the functional characterization of an ever increasing number of multi-gene families identified by complete genome analysis.