Amino Acid Functions in Protein Structure

Amino Acid Functions in Protein Structure

    • Alanine‘s role in structure: Alanine is not particularly hydrophobic and is non-polar. However, it contains a normal C-beta carbon, meaning that it is generally as hindered as other amino acids with respect to the conformations that the backbone can adopt. Alanine has a non-critical role in the protein contexts in general. The Alanine side chain is very non-reactive, and is thus rarely directly involved in protein function. However it can play a role in substrate recognition or specificity, particularly in interactions with other non-reactive atoms such as carbon.
Name Abbreviation
M.W.
pI
CAS Registry Number
Structure / formula
3D Structure
Alanine
89.09
6
56-41-7
ala
CH3-CH(NH2)-COOH
A
Ala A
    • Arginine is a positively charged, polar amino acid. Arginines prefer to be on the outside of proteins. It most prefers to substitute for the other positively charged amino acid Lysine. Arginine frequently plays an important role in structure. The part of the side chain nearest to the backbone is long, carbon containing and hydrophobic, whereas the end of the side chain is positively charged. So Arginines are usually at where part of the side-chain is buried, and only the charged portion is on the outside of the protein. Arginines are also frequently involved in salt-bridges, where they pair with a negatively charged amino acid (such as Aspartate) to create stabilizing hydrogen bonds, that can be important for protein stability.
Arginine
174.2
11.15
74-79-3
arg
HN=C(NH2)-NH-(CH2)3-CH(NH2)-COOH
R
Arg R
    • Asparagine is a polar amino acid. It prefers to substitute for other polar residues like Aspartate in particular. They differ only in that it contains an oxygen in place of the amino group in Asparagine. Asparagine are generally on the surface of proteins, exposed to an aqueous environment. Asparagines are frequently involved in protein active or binding sites. The polar side-chain is good for interactions with other polar or charged atoms.
Asparagine
132.12
5.41
5794-13-8
asn
H2N-CO-CH2-CH(NH2)-COOH
N
Asn N
    • Aspartate (or Aspartic acid) is a negatively charged, polar amino acid. It prefers to substitute for the other negatively charged amino acid like Glutamate and Asparginine. Asparagine are generally on the surface of proteins. When buried within the protein, Aspartates (and Glutamtes) are frequently involved in salt-bridges. They pair with a positively charged amino acid (such as Arginine) to create stabilizing hydrogen bonds, that can be important for protein stability. Aspartates are quite frequently involved in protein active or binding sites. The negative charge can interact with positively charged non-protein atoms, such as cations like zinc. Aspartate has a shorter side-chain than the very similar Glutamate meaning that is slightly more rigid within protein structures. This gives it a slightly stronger preference to be involved in protein active sites. Probably the most famous example of Asparate being involved in an active site is found within Serine proteases such as Trypsin, where it functions in the classical Asp-His-Ser catalytic triad.
Aspartic acid
133.1
2.77
56-84-8
asp
HOOC-CH2-CH(NH2)-COOH
D
Asp D
    • Cysteine shows no preference generally for substituting with any other amino acid. The role of Cysteines in structure is very dependent on the cellular location of the protein in which they are contained. Within extracellular proteins, cysteines are frequently involved in disulphide bonds, where pairs of cysteines are oxidized to form a covalent bond. These bonds serve mostly to stabilize the protein structure, and the structure of many extracellular proteins is almost entirely determined by the topology of multiple disulphide bonds. The reducing environment inside cells makes the formation of disulphide bonds very unlikely. Disulphides are also rare within the membrane, though membrane proteins can contain disulphide bonds within extracellular domains. In the intracellular environment Cysteines can still play a key structural role. Their sulfydryl side-chain is excellent for binding to metals, such as zinc, meaning the Cysteines (and other amino acids such as Histidines) are very common in metal binding motifs such as zinc fingers. Cysteines are also very common in protein active and binding sites. Binding to metals can also be important in enzymatic functions (e.g. metal proteases). Cysteine can also function as a nucleophile (i.e. the reactive centre of an enzyme). Probably the best known example of this occurs within the Cysteine proteases, such as caspases, or papains, where Cysteine is the key catalytic residue, being helped by a Histidine and an Asparagine.
Cysteine
121.15
5.02
52-90-4
cys
HS-CH2-CH(NH2)-COOH
C
Cys C
    • Glutamine is a polar amino acid. It prefers to substitute for other polar residues, in particular Glutamate, which differs only in that it contains an oxygen in place of the amino group in Glutamine. Being polar, Glutamine prefers generally to be on the surface of proteins, exposed to an aqueous environment. Glutamines are quite frequently involved in protein active or binding sites. The polar side-chain is good for interactions with other polar or charged atoms.
Glutamine
146.15
5.65
56-85-9
gln
H2N-CO-(CH2)2-CH(NH2)-COOH
Q
Gln Q
    • Glutamate (or Glutamic acid) is a negatively charged, polar amino acid. It prefers to substitute for the other negatively charged (and very similar) amino acid Aspartate, though it can also substitute with other polar amino acids, in particular Glutamine, which differs only in that it contains an amino group in place of one of the oxygens found in Glutamate (and thus also lacks a negative charge). Being charged and polar, Glutamates, prefer generally to be on the surface of proteins, exposed to an aqueous environment. When buried within the protein Glutamates (and Aspartates) are frequently involved in salt-bridges, where they pair with a positively charged amino acid to create stabilising hydrogen bonds, that can be important for protein stability. Glutamates are quite frequently involved in protein active or binding sites. The negative charge means that they can interact with positively charged non-protein atoms, such as cations like zinc. In certain cases, they can also perform a similar role to Aspartate, in the catalytic site of proteins such as proteases or lipases.
Glutamic acid
147.13
3.22
56-86-0
glu
HOOC-(CH2)2-CH(NH2)-COOH
E
Glu E
    • Glycine generally prefers to substitute with other small amino acids. Glycine is a very unique amino acid in that in contains a hydrogen rather than a carbon as its side chain. So Glycine has more conformational flexibility. Glycine can reside in parts of protein structures that are too tight or small to all other amino acids. Glycine can bind to phosphates due to its hydrogen side chain. If a conserved glycine was changed to any other amino acid, it will cause impacts to the protein structure and stability.
Glycine
75.07
5.97
56-40-6
gly
NH2-CH2-COOH
G
Gly G
    • Histidine is a polar amino acid. Histidine has a pKa near to that of physiological pH. So it is relatively easy to move protons on and off of the side chain, that is to change the side chain from neutral to positive charge. Histidines’ flexibility is rather ambiguous about whether it prefers to be buried in the protein core, or exposed to solvent. Histidine is an ideal residue for protein functional centres. Histidines are the most common amino acids in protein active or binding sites. They are very common in metal binding sites (e.g. zinc), often acting together with Cysteines or other amino acids.
Histidine
155.16
7.47
71-00-1
N=C-NH-C=C-CH2-CH(NH2)-COOH
H
|________|
His H
his
    • Isoleucine is an aliphatic, hydrophobic, amino acid. Isoleucine prefers to be buried in protein hydrophobic cores. Like Valine, and Threonine, Isoleucine is C-beta branched. Whereas most amino acids contain only one non-hydrogen substituent attached to their C-beta carbon, these three amino acids contain two. So these amino acids are more restricted in the conformations the main-chain can adopt. It is more difficult for these amino acids to adopt an alpha-helical conformation, though it is easy and even preferred for them to lie within beta-sheets. The Isoleucine side chain is very non-reactive, and is rarely directly involved in protein function, though it can play a role in substrate recognition. In particular, hydrophobic amino acids can be involved in binding/recognition of hydrophobic ligands such as lipids.
Isoleucine
131.17
5.94
73-32-5
ile
CH3-CH2-CH(CH3)-CH(NH2)-COOH
I
Ile I
    • Leucine is an aliphatic, hydrophobic, amino acid. Leucine prefers to be buried in protein hydrophobic cores. It also shows a preference for being within alpha helices than in beta strands. The Leucine side chain is very non-reactive, and is thus rarely directly involved in protein function, though it can play a role in substrate recognition. In particular, hydrophobic amino acids can be involved in binding/recognition of hydrophobic ligands such as lipids.
Leucine
131.17
5.98
61-90-5
leu
(CH3)2-CH-CH2-CH(NH2)-COOH
L
Leu L
    • Lysine is a positively charged, polar amino acid. Lysine frequently plays an important role in structure. The side chain nearest to the backbone is long, carbon containing and hydrophobic, whereas the end of the side chain is positively charged. Lysines are usually located where part of the side-chain is buried, and only the charged portion is on the outside of the protein. Generally Lysines prefer to be on the outside of proteins. Lysines are also frequently involved in salt-bridges, where they pair with a negatively charged amino acid (such as Aspartate) to create stabilizing hydrogen bonds, that can be important for protein stability. Lysines are quite frequent in protein active or binding sites. Lysine contains a positively charged amino on its side-chain that is sometimes involved in forming hydrogen bonds with negatively charged non-protein atoms (e.g. anions or carboxylate groups).
Lysine
146.19
9.59
39665-12-8
lys
H2N-(CH2)4-CH(NH2)-COOH
K
Lys K
    • Methionine is a hydrophobic amino acid. Methionine prefers to be buried in protein hydrophobic cores. The Methionine side chain is fairly non-reactive, and is thus rarely directly involved in protein function. Like other hydrophobic amino acids, it can play a role in binding/recognition of hydrophobic ligands such as lipids. Unlike the proper aliphatic amino acids, Methionine contains a sulphur atom, that can be involved in binding to atoms such as metals. However, whereas the sulphur atom in Cysteine is connected to a hydrogen atom, making it quite reactive, Methionines’ sulphur is connected to a methyl group. This means that the roles that Methionine can play in protein function are much more limited.
Methionine
149.21
5.74
63-68-3
met
CH3-S-(CH2)2-CH(NH2)-COOH
M
Met M
    • Phenylalanine is an aromatic, hydrophobic, amino acid. It particularly prefers to exchange with Tyrosine, which differs only in that it contains a hydroxyl group in place of the ortho hyddrogen on the benzene ring. Phenylalanine prefers to be buried in protein hydrophobic cores. The Phenylalanine side chain is fairly non-reactive, and is thus rarely directly involved in protein function, though it can play a role in substrate recognition. In particular, hydrophobic amino acids can be involved in binding/recognition of hydrophobic ligands such as lipids. Aromatic residues can also be involved in interactions with non-protein ligands that themselves contain aromatic groups via stacking interactions. Phenylalanine and other aromatic amino acids can be involved in binding to poly-proline containing peptides, for example, in SH3 or WW domains.
Phenylalanine
165.19
5.48
63-91-2
phe
Ph-CH2-CH(NH2)-COOH
F
Phe F
    • Proline is the only amino acid where the side chain is connected to the protein backbone twice, forming a five-membered nitrogen-containing ring. This makes Proline an imino acid since it contains an NH2+ rather than an NH3+ group. So Proline is unable to occupy many of the main chain conformations easily adopted by all other amino acids. In this sense, it can be considered to be an opposite of Glycine, which can adopt many more main-chain conformations. Proline can often be found in very tight turns in protein structures where the polypeptide chain must change direction. It can also function to introduce kinks into alpha helices, since it is unable to adopt a normal helical conformation. Prolines are usually be found on the protein surface. Proline plays important roles in molecular recognition, particularly in intracellular signaling. Domains such as WW and SH3 bind to specific proline containing peptides that are key parts of many signaling cascades. The Proline side chain is very non-reactive. Thus, together with its difficulty in adopting many protein main-chain conformations, it is very rarely involved in protein active or binding sites.
Proline
115.13
6.3
147-85-3
NH-(CH2)3-CH-COOH
P
|_________|
Pro P
pro
    • Serine is a slightly polar amino acid. Serine can reside both within the interior of a protein, or on the protein surface. Its small size means that it is relatively common within tight turns on the protein surface, where it is possible for the Serine side-chain hydroxyl oxygen to form a hydrogen bond with the protein backbone, effectively mimicking Proline. Serines are quite common in protein functional centers. The hydroxyl group is fairly reactive, being able to form hydrogen bonds with a variety of polar substrates. A common role for Serines (and Threonines and Tyrosines) within intracellular proteins is phosphorylation. Protein kinases frequently attach phosphates to Serines in order to fascilitate the signal transduction process. Serine can often be replaced by Threonine, but is unlikely to be replaced by Tyrosine, as the enzymes that catalyse the reactions (i.e. the protein kinases) are highly specific (i.e. Tyrosine kinases generally do not work on Serines/Threonines and vice versa).
Serine
105.09
5.68
56-45-1
ser
HO-CH2-CH(NH2)-COOH
S
Ser S
    • Threonine can reside both within the interior of a protein, or on the protein surface.Threonine is more restricted in the conformations the main-chain can adopt. It is more difficult for this amino acids to adopt an alpha-helical conformation, though it is easy and even preferred for them to lie within beta-sheets. Threonines are quite common in protein functional centers. The hydroxyl group is fairly reactive, being able to form hydrogen bonds with a variety of polar substrates. A common role for Threonines (and Serines and Tyrosines) within intracellular proteins is phosphorylation. Protein kinases frequently attach phosphates to Threonines in order to facilitate the signal transduction process. Threonine can often be replaced by Serine, but is unlikely to be replaced by Tyrosine, as the enzymes that catalyse the reactions are highly specific.
Threonine
119.12
5.64
72-19-5
thr
CH3-CH(OH)-CH(NH2)-COOH
T
Thr T
    • Tryptophan is an aromatic, hydrophobic, amino acid. Tryptophan prefers to be buried in protein hydrophobic cores. Tryptophan is involved in stacking interactions with other aromatic side-chains. As it contains a non-carbon atom (nitrogen) in the aromatic ring system, Tryptophan is more reactive than Phenylalanine though it is less reactive than Tyrosine. Like other aromatic amino acids, Tryptophan can be involved in interactions with non-protein ligands that themselves contain aromatic groups via stacking interactions. Tryptophan and other aromatic amino acids can be involved in binding to poly-proline containing peptides, for example, in SH3 or WW domains.
Tryptophan
204.23
5.89
73-22-3
Ph-NH-CH-C-CH2-CH(NH2)-COOH
W
|_______|
Trp W
trp
    • Tyrosine is an aromatic, partially hydrophobic, amino acid. Tyrosine prefers to be buried in protein hydrophobic cores. Tyrosine is involved in stacking interactions with other aromatic side-chains. Unlike the very similar Phenylalanine, Tyrosine contains a reactive hydroxyl group, thus making it much more likely to be involved in interactions with non protein atoms. Tryptophan and other aromatic amino acids can be involved in binding to poly-proline containing peptides, for example, in SH3 or WW domains. A common role for Tyrosines (and Serines and Threonines) within intracellular proteins is phosphorylation. Protein kinases frequently attach phosphates to Tyrosines in order to facilitate the signal transduction process. Tyrosine will rarely substitute for Serine or Threonine.
Tyrosine
181.19
5.66
60-18-4
tyr
HO-p-Ph-CH2-CH(NH2)-COOH
Y
Tyr Y
    • Valine is an aliphatic, hydrophobic, amino acid. Valine prefers to be buried in protein hydrophobic cores. The Valine side chain is very non-reactive, and is thus rarely directly involved in protein function, though it can play a role in substrate recognition. In particular, hydrophobic amino acids can be involved in binding/recognition of hydrophobic ligands such as lipids.
Valine
117.15
5.96
72-18-4
val
CH3-CH(CH2)-CH(NH2)-COOH
V

Methods for Detecting Protein Phosphorylation

Radiolabel studies suggest that approximately 30% of proteins in eukaryotic cells are subject to phosphorylation. A classical method of measuring protein phosphorylation involves 2D gel electrophoresis or incubation of cells with radiolabeled 32P-orthophosphate, the generation of cellular extracts, separation of proteins by SDS-PAGE, and then exposure to film. These methods are labor-intensive and requires radioisotopes. The following summary provides a brief description of several methodologies currently used to assess phosphorylation. Kinase Activity Assays Kinase activity is usually measured in vitro by incubating the immunoprecipitated kinase with a substrate in the presence of ATP. Measurement of the phosphorylated substrate can be assessed by reporter systems including colorimetric, radioactive, or fluorometric detection. Phospho-Specific Antibody Development The development of phosphorylation-dependent antibodies has been used by many researchers. LifeTein developed many phospho-specific antibodies for researchers. Phospho-specific peptides representing the amino acid sequence surrounding the phosphorylation site of the target protein were first synthesized and then conjugated to keyhole limpet hemocyanin (KLH) for immunization. The immune sera will then be applied to a peptide affinity column to generate a highly specific immunoreagent. The successful detection is dependent on the specificity and affinity of the antibody for the phospho-protein of interest. Western Blot Many phospho-specific antibodies are very sensitive and can readily detect the phosphorylated protein in a routine sample. Both chemiluminescent and colorimetric detection methods are common, and molecular weight markers are also generally used to provide information about protein mass. Enzyme-Linked Immunosorbent Assay (ELISA) ELISAs generally provide an indirect measurement of kinase activity and is more quantitative than Western blotting. The phospho-specific ELISA technique can easily quantify the results by utilizing a calibrated standard. Using of two antibodies specific for the target protein employed together in the sandwich format will render high specificity. In addition, the microplate-based format of ELISAs allows for high throughput and smaller sample volumes and the detection of low abundance proteins. Cell-Based ELISA Analyzing protein phosphorylation within intact cells may more accurately represent the status of specific signaling networks. Usually phospho-specific antibodies are used to assess phosphorylation status using fluorometric or colorimetric detection systems. Intracellular Flow Cytometry and ICC/IHC Flow cytometry is advantageous because it allows for rapid, quantitative, single cell analysis.Cells are usually stimulated and fixed with formaldehyde or paraformaldehyde to cross-link the phospho-proteins and stabilize them for analysis. The fixed cells must then be permeabilized to allow for entry of phospho-specific antibodies into the cells. Mass Spectrometry Mass spectrometry (MS) techniques are useful tools for identification of phospho-proteins and phosphopeptides and sequencing of the phosphorylated residues. MS can be used with excellent sensitivity and resolution to identify a single protein or peptide. Although signals from phosphopeptides are generally weaker, new technologies have beed developed to enrich the MS signals. The enrichment strategies include immobilized metal affinity chromatography, phosphospecific antibody enrichment, chemical-modification-based methods and replacement of the phosphate group with biotinylated moieties. Multi-Analyte Profiling Multi-Analyte profiling involves the use of phospho-specific antibodies and include microplate-based and membrane-based detection formats. These assays provide more data while requiring very little sample volume. However these assays are less sensitive than the conventional methods due to potential antibody cross-reactivity.

Phospho-specific antibodies by LifeTein published in Nature

Jia Shen. et al. EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2. Nature 497, 383–387 (16 May 2013), doi:10.1038/nature12080 LifeTein helped designed and synthesized a series of phosphorylated peptides. Then the peptides were used for phospho-specific antibody productions. The phospo-specific antibodies by LifeTein were confirmed to react with the epidermal growth factor receptor (EGFR). The Hung’s lab showed that AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells. … The following peptides were chemically synthesized for antibody production in mice (Lifetein Conc.), Elisa verification (LifeteinConc.) and peptide competition assay in immunohistochemistry (IHC)… Supplementary information

TFA removal

Trifluoroacetic acid (TFA) is commonly used to release synthesized peptides from solid-phase resins. TFA or acetate is also used during reversed-phase HPLC purification of peptides. Residual TFA or fluoride are toxic and undesirable in peptides intended for preclinical and clinical studies. TFA can be very problematic. Hence we can either get rid of the TFA salts or avoid them from the beginning. LifeTein provides TFA removal service to avoid the toxicity to cells for the clinical purpose. TFA is a strong acid. It will protonate any amino group. The same goes for HCl. One technique is to do an anion exchange on the same reversed phase HPLC on which the peptide was purified. Load the peptide on the column. Hold it on the top while washing it with enough amount of acetic acid buffer, and then elute it out with an aqueous acetic acid/acetonitrile gradient. After freeze drying, the counterion TFA will be exchanged. The technique relies on the hydrophobicity of the peptide. An very hydrophilic peptide will require a proper anion exchange resin. LifeTein uses a special reverse phase C-18 material that provides good separations without TFA. The separation of TFA, acetate, and fluoride uses a stationary phase functionalized with alkyl quaternary ammonium groups. Effluent from the analytical column is passed through a suppressor that reduces the total background conductance of the eluent and increases the electrical conductance of the analyte ions. With suppressed conductivity, signal-to-noise ratios are improved approximately 50-fold compared to nonsuppressed conductivity. http://lifetein.com/TFA_Removal_Peptide_Synthesis_Services.html

Co-Immunoprecipitation, Co-IP

One of the most commonly used methods for verification of protein–protein interactions is Co-immunoprecipitation (Co-IP). Co-IP is to identify physiologically relevant protein-protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein. In a typical experiment, bait complexes are captured from a sample such as a cell lysate using a specific antibody. The immune complex is then captured and immobilized using protein A or protein G covalently attached to sepharose beads. After washing of the beads, the antibody, the bait and proteins associated to the bait are eluted from the support. The bound proteins can then be identified by MS or by immunoblotting. Performing a co-IP reaction and identifying physiological protein-protein interactions can be difficult and generate significant background. This is because of the nature of the interaction, nonspecific binding to IP components and antibody contamination that may mask detection. It is therefore important to conduct parallel negative controls. Co-IP experiments can be carried out from cell-lines or tissues expressing their endogenous proteins, cells transfected with a plasmid encoding a tagged bait protein, or cells transfected with tagged versions of two putative interaction partners. Usually highly specific antibodies are required for Co-IP. However an antibody directed against the tag (instead of against the bait protein) can then be used if cells transfected with a plasmid encoding a tagged bait protein. The epitope-tagged proteins can often be eluted by incubation with competing peptides. This specific elution often reduces the amount of contaminating proteins in the eluate. Co-IP Optimization
  • It is important to optimize the experiments to improve detection. Many protein interactions will remain intact after lysis using standard non-denaturing lysis buffers with low ionic strength (i.e., <120mM NaCl) that contain non-ionic detergents (NP-40 and Triton X-100). They are less likely to disrupt protein-protein interactions. The most generally effective, nondenaturing elution buffer for protein affinity purification methods is 0.1 M glycine at pH 2.5 to 3. The low pH condition dissociates most antibody-antigen interactions, as well as the antibody-Protein A/G interaction.
  • Sonication or vortexing the bead-bound immune complexes should be avoided to prevent the disruption of the protein-protein interaction(s) of the target complex. The samples should be handled gently to prevent the loss of bound complex proteins.
  • High background from nonspecificc interactions may be avoided by washing the immune complexes thoroughly. Preclearing the lysates is important to remove potentially reactive components. Preclearing by incubating the sample with a nonspecific antibody from the same species as the IP antibody. Any nonspecific immune complexes will form and be immobilized to the beaded support. The nonspecific lysate products will be removed by this preclearing step so that they will not co-purify with the target antigen in the actual IP experiment. Titrating the salt concentration from 120 to 1000mM or primary antibody for the maximized signal:noise ratio will be useful to reduce the background.
  • The presence of the co-eluted antibody light and heavy chains (25 and 50kDa bands in reducing SDS-PAGE gels, respectively) in the sample can mask the results. Be sure to elute the antigen under non-denaturing conditions. Otherwise, the denatured antibody fragments will be eluted with the antigen. Crosslinking antibody to Protein A/G-coated beads or covalently binding antibody directly to treated beads are very effective ways to circumvent antibody contamination. Utilizing strong biotin streptavidin reation is another way for clean Co-IP results. The IP antibody is biotinylated and the beads are coated with streptavidin. The immune complexes are captured by the beads. The antibody is not eluted from the beads when mild conditions are used to release the target antigen.

Public prtein-protein interaction databases After the initial screen for protein–protein interactions a list of potential targets has been generated. The next step is to search the literature to find out if there are any proteins in the list that has been detected by other investigators as binding partners to the bait protein.
DIP (http://dip.doe-mbi.ucla.edu/), IntAct (http://www.ebi.ac.uk/intact), MINT (http://mint.bio.uniroma2.it/mint/Welcome.do) MIPS (http://mips.gsf.de/genre/proj/mpact/) iHOP(http://www.ihop-net.org/UniPub/iHOP/) IntAct (http://www.ebi.ac.uk/intact)

Synthesis of multiple antigenic peptides:strategies and limitations

Synthesis of multiple antigenic peptides: strategies and limitations Dendrimeric platforms such as MAPs can be synthesized by solid phase method or by conjugation . Dendrimeric platforms such as MAPs can be synthesized either entirely by solid-phase methods (SPPS, direct approach) or by conjugation in solution of preformed, SPPS-made building blocks (indirect approach).
MAP peptide synthesis

MAP peptide synthesis

http://www.ncbi.nlm.nih.gov/pubmed/21391284 SPPS is the preferred method by LifeTein. The synthesis approach requires a branched poly-lysine core. Each branch is elongated into the corresponding epitope by stepwise SPPS. The disadvantage of this approach is that the synthetic errors could happened and cause microheterogeneity in the final materials. However the cost is lower and less time-consuming than the indirect approach. For very long linear peptides, it is more advantageous to use the SPPS method. The MAP synthesis may not always meet with success. The solubility of the peptide epitope can also become an issue and is difficult to predict for long epitopes. It is recommended to carefully design and analyze the linear epitope before MAP synthesis. Studies showed that synthesis with Ahx linker in the lysine core had better isolated yield. It is possible that the flexibilizing effect of Ahx helps in keeping peptide chains properly solvated during synthesis, preventing aggregation and hence increasing the amount of viable growing peptide sequences.  

How to Detect Small Peptides by SDS-PAGE?

Does your sample contain proteins of interest that are <20 kDa? Please download a protocol on how to detect synthetic peptides using SDS-PAGE. Tricine-SDS-PAGE is commonly used to separate proteins in the mass range of 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. It is indeed very difficult to see the small peptide by SDS-PAGE. Tris-tricine gel will give you a better resolution. If you just want to detect the peptide, Mass Spec is still the best way to confirm the peptide identity. Small peptide binds less Coomassie brilliant blue than larger protein. Thus smaller peptides are harder to detect by coomassie staining or silver staining. If you really want to see your peptide on the gel, you can try to load more samples. Changing the gel percentage won’t help much unless you think your peptide migrated out of the gel. You can increase the percentage of cross linker in the regular 17% gel. In addition increase the pH of your resolving gel to 9.5 as compared to your regular 8.8. Plus, the addition of urea (4-8M) helps sharpen bands. If you are going to use western, which is a way more sensitive detection method, please use Western instead of the gel staining. However the peptide may simply pass through the membrane. If you repeat the experiment, try to put two pieces of membrane and shorter time of transfer (less than 1 hour at 200 mA). 0.2um pore could be enough. You can get smaller pore but that shouldn’t be necessary. You may want to try semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus. A short 15 min transfer time works for most of the small peptides. If you can plan ahead and synthesize a control small peptide labeled with biotin, you can monitor the transfer process and its ability to bind the membrane with streptavidin-conjugated HRP. Please download this protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue staining, silver staining, and electroblotting. Download the Protocol
    • To download the protocol, click the download button below.
peptide synthesis service

Peptide-binding Characteristics

An old adage says: “Show me your friends, and I’ll know who you are.” In the same way, identifying the compounds capable of interacting with a specific protein can reveal its function. Synthetic peptides are one of the approaches for detecting protein interactions. An hsp70 (heat-shock protein of relative molecular mass 70K) can distinguish only unfolded forms of protein. To study the amino acid preferences, Gregory C. Flynn et. al. used the random-sequence peptides to fill the binding site of Binding immunoglobulin protein (BiP). It was found that the binding site of BiP shows considerable specificity.
  • Peptide length: Peptides that are 7 amino acids in length can bind to the hsp70 family. Other peptides 6-10 residues in length can usually bind reasonably well. Longer peptides do not increase the binding activity.
  • Amino acid preferences: Peptide chains capable of binding to BiP complexes consistently show enrichment in the aliphatic amino acids, such as Glycine, Alanine, Valine, Leucine, Isoleucine, and Proline, at all positions.
    • Leucine has the highest abundance in BiP complexes.
    • Amino acid with unbranched side chains (such as Methionine) and side chains that branch away (such as Leucine) tend to bind to the BiP complexes. This is probably because side chain flexibility and hydrophobicity both facilitate binding.
    • Even though they are the most hydrophobic, the aromatics (Phenylalanine, Tyrosine, and Tyrptophan) are less important to binding because they do not have flexible side chains.
    • Random 7-mers containing an average of 1.6 aliphatic residues binds productively to BiP.
Reference: Gregory C. Flynn, Jan Pohl, Mark T. Flocco & James E. Rothman. Peptide-binding specificity of the molecular chaperone BiP. 24 October 1991, Nature 353, 726-730 doi:10.1038/353726a0. http://www.lifetein.com/Protein-peptide-interactions.html

The Structural Basis of Peptide-Protein Binding Strategies

peptide protein binding strategy

Peptide protein binding strategies: Structure, Volume 18, Issue 2, 188-199, 10 February 2010

  Highlights
  • Most peptides do not induce conformational changes on their partner upon binding
  • Peptide-protein interfaces are better packed and contain more hydrogen bonds
  • Binding is mediated by peptide hotspots that contribute most of the binding energy
  • Peptides tend to bind in the largest pockets or holes on the protein surface
Read more from here. LifeTein is pleased to offer a free, comprehensive web-based peptide analysis tool. This tool will allow your research team to overcome common difficulties inherent in protein analysis and peptide antigen design.
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Peptide analysis tool-LifeTein

D-amino acid peptides to resist common proteases

Peptides that are at least partially made of D-amino acids have shown strong resistance to proteolytic degradation.
D amino acid peptide with high stability

D amino acid peptide with high stability

See more details from here: http://lifetein.com/Peptide-Synthesis-D-Amino-Acid.html Reference: http://www.pnas.org/content/102/2/413.full.pdf+html Google+