Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns

To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips.     

Fusion order controls expression level and activity of elastin‐like polypeptide fusion proteins

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin‐like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C‐terminus of the target protein (protein‐ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP‐protein constructs have a lower specific activity than protein‐ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein‐ELP and ELP‐protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N‐terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C‐terminal end of the protein.     

Molecular rearrangements of the Kv2.1 potassium channel termini associated with voltage gating

The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFP(N)-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.     

Protein body formation in leaves of Nicotiana benthamiana: a concentration‐dependent mechanism influenced by the presence of fusion tags

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin‐like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP‐ and HFBI‐induced PBs and showed that ELP‐induced PBs are larger than HFBI‐induced PBs. The size of ELP‐ and HFBI‐induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration‐dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER‐resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin‐10 by co‐expression with PB‐inducing proteins.     

Unexpected multivalent display of proteins by temperature triggered self-assembly of elastin-like polypeptide block copolymers

We report herein the unexpected temperature triggered self-assembly of proteins fused to thermally responsive elastin-like polypeptides (ELPs) into spherical micelles. A set of six ELP block copolymers (ELP(BC)) differing in hydrophilic and hydrophobic block lengths were genetically fused to two single domain proteins, thioredoxin (Trx) and a fibronectin type III domain (Fn3) that binds the α(v)β(3) integrin. The self-assembly of these protein-ELP(BC) fusions as a function of temperature was investigated by UV spectroscopy, light scattering, and cryo-TEM. Self-assembly of the ELP(BC) was unexpectedly retained upon fusion to the two proteins, resulting in the formation of spherical micelles with a hydrodynamic radius that ranged from 24 to 37 nm, depending on the protein and ELP(BC). Cryo-TEM images confirmed the formation of spherical particles with a size that was consistent with that measured by light scattering. The bioactivity of Fn3 was retained when presented by the ELP(BC) micelles, as indicated by the enhanced uptake of the Fn3-decorated ELP(BC) micelles in comparison to the unimer by cells that overexpress the α(v)β(3) integrin. The fusion of single domain proteins to ELP(BC)s may provide a ubiquitous platform for the multivalent presentation of proteins.     

Quaternary structure is critical for protein display on capsid-like particles (CLPs): efficient generation of hepatitis B virus CLPs presenting monomeric but not dimeric and tetrameric fluorescent proteins

Self-organizing assemblies such as viral capsids may be used as symmetrical molecular platforms for the display of heterologous sequences, with applications ranging from vaccines to structural studies. The 183-amino-acid hepatitis B virus (HBV) core protein assembles spontaneously into icosahedral capsid-like particles (CLPs). The most exposed, and most immunogenic, substructure on the CLPs is a small loop that connects two long antiparallel alpha-helices which act as dimerization interface. Ninety (90) or 120 dimers multimerize into the capsid; the four-helix bundles formed by the dimers protrude as spikes from the surface. We recently demonstrated that the entire enhanced green fluorescent protein (eGFP) can be inserted into this loop, yielding CLPs that natively displayed eGFP on their surface. The central location of the insertion site requires that any insert be fixed to the carrier via both termini, with corresponding restrictions regarding insert size and structure. eGFP obviously satisfied these criteria but, surprisingly, all attempts to produce CLPs with the isostructural red fluorescent proteins DsRed1, DsRed2, and HcRed failed. Suspecting their oligomerization tendency to be responsible, we generated fusions containing instead monomeric yellow, cyan, and red fluorescent proteins (mYFP, mCFP and mRFP1). This strongly increased the yields of YFP and CFP-CLPs, and it allowed for the first time to efficiently generate red fluorescent CLPs. Hence insert quaternary structure is a highly critical factor for CLP assembly. These data have important implications for the rational design of self-assembling fusion proteins.     

Global and local dynamics of the U1A polyadenylation inhibition element (PIE) RNA and PIE RNA-U1A complexes

The structure and dynamics of the polyadenylation inhibition element (PIE) RNA, free and bound to the U1A protein, have been examined using time-resolved FRET and 2-aminopurine (2AP) fluorescence. This regulatory RNA, located at the 3' end of the U1A pre-mRNA, adopts a U-shaped structure, with binding sites for a single U1A protein at each bend (box 1 and box 2). The distance between the termini of the arms of the RNA is sensitive to its three-dimensional structure. Using Cy3/Cy5 FRET efficiency to monitor binding of Mg(2+), we show that the PIE RNA binds two Mg(2+) ions, which results in a restriction of its distance distribution of conformations. Local RNA structure probing using 2AP fluorescence shows that the structure of box 2 changes in response to Mg(2+) binding, thus tentatively locating the ion binding sites. Steady-state FRET data show that the distance R between the termini of the PIE RNA stems decreases from 66 A in the free RNA, to 58 A when N-terminal RNA binding domains (RBD1) of U1A are bound, and to 53 A when U1A proteins bind. However, anisotropy measurements indicate that both Cy3 and Cy5 stack on the ends of the RNA. To examine the consequences of the restricted motion of the fluorophores, FRET data are analyzed using two different models of motion and then compared to analogous data from the Cy3/fluorescein FRET pair. We conclude that the error introduced into distance calculations by stacking of the dyes is within the error of our measurements. Distance distributions of the RNA structures show that the intramolecular distance between the arms of the PIE RNA varies on the time scale of the fluorescence measurements; the mean distance is dependent on protein binding, but the breadth of the distributions indicates that the RNA retains structural heterogeneity.     

Multidomain initiation factor 2 from Thermus thermophilus consists of the individual autonomous domains

The three-dimensional chalice-like crystal structure of initiation factor 2 IF2/eIF5B from Methanobacterium thermoautotrophicum represents a novel fold and domain architecture in which the N-terminal G domain and the C-terminal C domain are separated by an approximately 40 A alpha-helix. Homologous Thermus thermophilus initiation factor 2 (IF2wt), G (IF2G), and C (IF2C) domains were successfully overexpressed and purified which enabled us to perform a thermodynamic analysis and to asses the role of the domain architecture in this atypical fold. Circular dichroism in the far-UV region demonstrated that the proteins are well-folded and that the secondary structure content resembles that of IF2 from M. thermoautotrophicum. IF2wt and IF2G are monomeric proteins, while IF2C has a tendency to form dimeric species as shown by sedimentation velocity studies on analytical ultracentrifugation and differential scanning calorimetry scan analysis. Thermal denaturation studies of multidomain IF2wt reveals an exceptionally high reversibility (>90%) of the transition with a melting temperature of 94.5 degrees C. Melting temperature of IF2wt may be further increased in the presence of its physiological ligand GDP and the GTP analogue, GppNHp. The high reversibility of denaturation is achieved by the modular structure of the protein and by the high reversibility of the thermal denaturation of IF2G. On the other hand, hydrophobic IF2C aggregates during the thermal transition, and the aggregation is suppressed by guanidine hydrochloride. Isothermal denaturation demonstrates that both IF2G and IF2C have comparable stabilities of 46 and 33 kJ/mol, respectively. The apparent cooperative unfolding of the full-length protein has an unusually small denaturant m value. This together with the phase diagram method of analysis indicates the presence of intermediate(s) due to the independent unfolding of IF2G and IF2C. Despite an absence of apparent interactions between the domains in vitro, IF2G plays a role in IF2C reversibility in thermal denaturation. In conclusion, interactions between the domains of folded IF2wt in vivo are likely mediated by their alpha-helix connection and/or by a conformational change on the ribosome.     

Design of the linkers which effectively separate domains of a bifunctional fusion protein

With the aim of separating the domains of a bifunctional fusion protein, the ability of several lengths of helix-forming peptides to separate two weakly interacting beta-can domains was compared with that of flexible linkers or of a three alpha-helices bundle domain. We introduced helix-forming peptide linkers A(EAAAK)nA (n = 2-5) between two green fluorescent protein variants, EBFP and EGFP, and investigated their spectral properties. The fluorescence resonance energy transfer from EBFP to EGFP decreased as the length of the linkers increased. The circular dichroism spectra analysis suggested that the linkers form an alpha-helix and the alpha-helical contents increased as the length of the linkers increased. The results clearly suggested the ability of the helical linkers to control the distance and reduce the interference between the domains. This 'linker engineering' may open a way to the rational design of linkers which maximize the multiple functions of fusion proteins or de novo multi-domain proteins.     

A critical assessment of the secondary structure alpha-helices and their termini in proteins

Secondary structure prediction from amino acid sequence is a key component of protein structure prediction, with current accuracy at approximately 75%. We analysed two state-of-the-art secondary structure prediction methods, PHD and JPRED, comparing predictions with secondary structure assigned by the algorithms DSSP and STRIDE. The specific focus of our study was alpha-helix N-termini, as empirical free energy scales are available for residue preferences at N-terminal positions. Although these prediction methods perform well in general at predicting the alpha-helical locations and length distributions in proteins, they perform less well at predicting the correct helical termini. For example, although most predicted alpha-helices overlap a real alpha-helix (with relatively few completely missed or extra predicted helices), only one-third of JPRED and PHD predictions correctly identify the N-terminus. Analysis of neighbouring N-terminal sequences to predicted helical N-termini shows that the correct N-terminus is often within one or two residues. More importantly, the true N-terminal motif is, on average, more favourable as judged by our experimentally measured free energies. This suggests a simple, but powerful, strategy to improve secondary structure prediction using empirically derived energies to adjust the predicted output to a more favourable N-terminal sequence.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Structural analysis of the phototactic transducer protein HtrII linker region from Natronomonas pharaonis

Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 microM. The 3D structure of this fragment was determined by solution NMR, and only one alpha-helix was found between two HAMP domains of the linker region. This alpha-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The alpha-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream.     

DNA-directed assembly of supramolecular fluorescent protein energy transfer systems

Fluorescent proteins with a wide variety of physicochemical properties have evolved in the past few years. The use of these proteins for applications in biomolecular nanosciences requires their precise positioning at the nanometer length scale. To address this challenge, we report here on the self-organization of DNA-tagged fluorescent probes to construct a set of photofunctional supramolecular complexes which include the enhanced yellow fluorescent protein (EYFP). The optical functionality is based on the strongly distance dependent fluorescence resonance energy transfer (FRET), occurring between the donor (EYFP) and an acceptor fluorophore, i.e., the fluorescent dye Atto647. The photophysical properties of four bimolecular FRET complexes, each possessing a well-defined donor-acceptor distance defined by the length of the interconnecting DNA backbone, are investigated by two-dimensional photoluminescence excitation spectroscopy (2D-PLE).     

Carboxy-terminal SEKDEL sequences retard but do not retain two secretory proteins in the endoplasmic reticulum

The sequence Ser-Glu-Lys-Asp-Glu-Leu (SEKDEL) has been shown to be a signal which leads to retention of at least two proteins in the endoplasmic reticulum of animal cells (Munro and Pelham, 1987). In this study we tested the function of this signal by appending it to two secretory proteins, rat growth hormone and the alpha subunit of human chorionic gonadotrophin (hCG-alpha). We used oligonucleotide-directed mutagenesis and expression to generate proteins with SEKDEL added to the exact COOH termini and then carried out a detailed analysis of their transport in monkey COS cells. We found that transport was not blocked for either protein, but rather that the half-time for secretion was increased about sixfold for both proteins. Analysis of oligosaccharide processing on hCG-alpha-SEKDEL and indirect immunofluorescence microscopy on cells expressing both proteins was consistent with a retardation of transport between the endoplasmic reticulum and the Golgi apparatus. A change in the last amino acid of the SEKDEL sequence from Leu to Val abolished the retardation almost completely, suggesting a highly specific interaction of the sequence with a receptor. A change in the first amino acid had little or no effect on retardation. We conclude that the SEKDEL signal can have strong effects on reducing the rate of protein exit from the endoplasmic reticulum without generating absolute retention. Presumably other features of protein structure must be important to generate absolute retention.     

Helix capping in the GCN4 leucine zipper.

Capping interactions associated with specific sequences at or near the ends of alpha-helices are important determinants of the stability of protein secondary and tertiary structure. We investigate here the role of the helix-capping motif Ser-X-X-Glu, a sequence that occurs frequently at the N termini of alpha helices in proteins, on the conformation and stability of the GCN4 leucine zipper. The 1.8 A resolution crystal structure of the capped molecule reveals distinct conformations, packing geometries and hydrogen-bonding networks at the amino terminus of the two helices in the leucine zipper dimer. The free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is -1.2 kcal/mol, evaluated from thermal unfolding experiments. A single cap thus contributes appreciably to stabilizing the terminated helix and thereby the native state. These results suggest that helix capping plays a further role in protein folding, providing a sensitive connector linking alpha-helix formation to the developing tertiary structure of a protein.     

Coil-to-helix transition and ligand release of Bombyx mori pheromone-binding protein

The transport of hydrophobic insect pheromones through the aqueous medium surrounding their receptors is assisted by pheromone-binding proteins (PBPs). The protein from the silkworm moth Bombyx mori, BmorPBP, exhibits a pH-dependent conformational change postulated to trigger the release of the pheromone bombykol to its receptor. At low pH, an alpha-helix occupies the same binding pocket that houses the pheromone in the BmorPBP-bombykol complex at high pH. We have determined the crystal structure of apo BmorPBP at a resolution of 2.3 angstroms and pH 7.5, which has surprisingly a structure similar to the A-form. These data suggest that BmorPBP undergoes a ligand-dependent conformational change in addition to the previously described pH-dependent conformational change. Analysis of the alpha-helix occupying the binding pocket reveals an amphipathic helix with three acidic residues along one face that are conserved among lepidopteran PBPs and may be involved in a conformational transition of BmorPBP at the receptor membrane.     

Side-chain structures in the first turn of the alpha-helix

The first three residues at the N terminus of the alpha-helix are called N1, N2 and N3. We surveyed 2102 alpha-helix N termini in 298 high-resolution, non-homologous protein crystal structures for N1, N2 and N3 amino acid and side-chain rotamer propensities and hydrogen-bonding patterns. We find strong structural preferences that are unique to these sites. The rotamer distributions as a function of amino acid identity and position in the helix are often explained in terms of hydrogen-bonding interactions to the free N1, N2 and N3 backbone NH groups. Notably, the "good N2" amino acid residues Gln, Glu, Asp, Asn, Ser, Thr and His preferentially form i, i or i,i+1 hydrogen bonds to the backbone, though this is hindered by good N-caps (Asp, Asn, Ser, Thr and Cys) that compete for these hydrogen bond donors. We find a number of specific side-chain to side-chain interactions between N1 and N2 or between the N-cap and N2 or N3, such as Arg(N-cap) to Asp(N2). The strong energetic and structural preferences found for N1, N2 and N3, which differ greatly from positions within helix interiors, suggest that these sites should be treated explicitly in any consideration of helical structure in peptides or proteins.     

A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling

We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "Azami-Green (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric green-emitting fluorescent protein comparable to EGFP.     

A novel approach of protein immobilization for protein chips using an oligo-cysteine tag

Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamond-like, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.     

NMR analysis of synthetic human serum albumin alpha-helix 28 identifies structural distortion upon amadori modification

The non-enzymatic reaction between reducing sugars and long-lived proteins in vivo results in the formation of glycation and advanced glycation end products, which alter the properties of proteins including charge, helicity, and their tendency to aggregate. Such protein modifications are linked with various pathologies associated with the general aging process such as Alzheimer disease and the long-term complications of diabetes. Although it has been suggested that glycation and advanced glycation end products altered protein structure and helicity, little structural data and information currently exist on whether or not glycation does indeed influence or change local protein secondary structure. We have addressed this problem using a model helical peptide system containing a di-lysine motif derived from human serum albumin. We have shown that, in the presence of 50 mm glucose and at 37 degrees C, one of the lysine residues in the di-lysine motif within this peptide is preferentially glycated. Using NMR analysis, we have confirmed that the synthetic peptide constituting this helix does indeed form a alpha-helix in solution in the presence of 30% trifluoroethanol. Glycation of the model peptide resulted in the distortion of the alpha-helix, forcing the region of the helix around the site of glycation to adopt a 3(10) helical structure. This is the first reported evidence that glycation can influence or change local protein secondary structure. The implications and biological significance of such structural changes on protein function are discussed.