Secondary Structure In Solution Research Articles

Structured and Unstructured Binding of an Intrinsically Disordered Protein as Revealed by Atomistic Simulations.

Intrinsically disordered proteins (IDPs) are a set of proteins that lack a definite secondary structure in solution. IDPs can acquire tertiary structure when bound to their partners; therefore, the recognition process must also involve protein folding. The nature of the transition state (TS), structured or unstructured, determines the binding mechanism. The characterization of the TS has become a major challenge for experimental techniques and molecular simulations approaches since diffusion, recognition, and binding is coupled to folding. In this work we present atomistic molecular dynamics (MD) simulations that sample the free energy surface of the coupled folding and binding of the transcription factor c-myb to the cotranscription factor CREB binding protein (CBP). This process has been recently studied and became a model to study IDPs. Despite the plethora of available information, we still do not know how c-myb binds to CBP. We performed a set of atomistic biased MD simulations running a total of 15.6 μs. Our results show that c-myb folds very fast upon binding to CBP with no unique pathway for binding. The process can proceed through both structured or unstructured TS's with similar probabilities. This finding reconciles previous seemingly different experimental results. We also performed Go-type coarse-grained MD of several structured and unstructured models that indicate that coupled folding and binding follows a native contact mechanism. To the best of our knowledge, this is the first atomistic MD simulation that samples the free energy surface of the coupled folding and binding processes of IDPs.

NMR backbone resonance assignment and solution secondary structure determination of human NSD1 and NSD2.

Proteins of the NSD family are histone-methyl transferases with critical functions in the regulation of chromatin structure and function. NSD1 and NSD2 are homologous proteins that function as epigenetic regulators of transcription through their abilities to catalyse histone methylation. Misregulation of NSD1 and NSD2 expression or mutations in their genes are linked to a number of human diseases such as Sotos syndrome, and cancers including acute myeloid leukemia, multiple myeloma, and lung cancer. The catalytic domain of both proteins contains a conserved SET domain which is involved in histone methylation. Here we report the backbone resonance assignments and secondary structure information of the catalytic domains of human NSD1 and NSD2.

Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis.

RNA modification is a post-transcriptional process by which certain nucleotides are altered after their initial incorporation into an RNA chain. Transfer RNAs (tRNAs) is the most heavily modified class of RNA molecules. These modifications expand the chemical and functional diversity of tRNAs and enhance their structural stability. To date, more than 100 modifications have been identified, the majority of which are specific from one domain of life. However, few modifications are extensively present in the three domains of life. Among those, the m(1)A nucleotide, which consists in the methylation at position 1 of the adenine aromatic ring, is found in tRNAs and ribosomal RNAs. In tRNAs, the m(1)A modification occurs at position 9, 14, 22, 57 and 58. The enzyme TrmK catalyzes the m(1)A formation at position 22. Here we report the backbone (1)H, (15)N and (13)C chemical shift assignments of TrmK from Bacillus subtilis obtained by heteronuclear multidimensional NMR spectroscopy as well as its secondary structure in solution as predicted by TALOS+. These assignments of TrmK pave the way for interaction studies with its tRNA substrates.

Folding of ortho-Phenylenes.

In nature, the folding of oligomers and polymers is used to generate complex three-dimensional structures, yielding macromolecules with diverse functions in catalysis, recognition, transport, and charge- and energy-transfer. Over the past 20-30 years, chemists have sought to replicate this strategy by developing new foldamers: oligomers that fold into well-defined secondary structures in solution. A wide array of abiotic foldamers have been developed, ranging from non-natural peptides to aromatics. The ortho-phenylenes represent a recent addition to the family of aromatic foldamers. Despite their structural simplicity (chains of benzenes connected at the ortho positions), it was not until 2010 that systematic studies of o-phenylenes showed that they reliably fold into helices in solution (and in the solid state). This conformational behavior is of fundamental interest: o-Arylene and o-heteroarylene structures are found embedded within many other systems, part of an emerging interest in sterically congested polyphenylenes. Further, o-phenylenes are increasingly straightforward to synthesize because of continuing developments in arene-arene coupling, the Asao-Yamamoto benzannulation, and benzyne polymerization. In this Account, we discuss the folding of o-phenylenes with emphasis on features that make them unique among aromatic foldamers. Interconversion between their different backbone conformers is slow on the NMR time scale around room temperature. The (1)H NMR spectra of oligomers can therefore be deconvoluted to give sets of chemical shifts for different folding states. The chemical shifts are both highly sensitive to conformation and readily predicted using ab initio methods, affording critical information about the conformational distribution. The picture that emerges is that o-phenylenes fold into helices with offset stacking between every third repeat unit. In general, misfolding occurs primarily at the oligomer termini (i.e., "frayed ends"). Because of their structural simplicity, the folding can be described by straightforward models. The overall population can be divided into two enantiomeric pools, with racemization and misfolding as two distinct processes. Examination of substituent effects on folding reveals that the determinant of the relative stability of different conformers is (offset) aromatic stacking interactions parallel to the helical axis. That is, the folding of o-phenylenes is analogous to that of α-helices, with aromatic stacking in place of hydrogen bonding. The folding propensity can be tuned using well-known substituent effects on aromatic stacking, with moderate electron-withdrawing substituents giving nearly perfect folding. The combination of a simple folding mechanism and readily characterized conformational populations makes o-phenylenes attractive structural motifs for incorporation into more-complex architectures, an important part of the next phase of foldamer research.

Destabilization of α-Helical Structure in Solution Improves Bactericidal Activity of Antimicrobial Peptides: Opposite Effects on Bacterial and Viral Targets.

We have previously examined the mechanism of antimicrobial peptides on the outer membrane of vaccinia virus. We show here that the formulation of peptides LL37 and magainin-2B amide in polysorbate 20 (Tween 20) results in greater reductions in virus titer than formulation without detergent, and the effect is replicated by substitution of polysorbate 20 with high-ionic-strength buffer. In contrast, formulation with polysorbate 20 or high-ionic-strength buffer has the opposite effect on bactericidal activity of both peptides, resulting in lesser reductions in titer for both Gram-positive and Gram-negative bacteria. Circular dichroism spectroscopy shows that the differential action of polysorbate 20 and salt on the virucidal and bactericidal activities correlates with the α-helical content of peptide secondary structure in solution, suggesting that the virucidal and bactericidal activities are mediated through distinct mechanisms. The correlation of a defined structural feature with differential activity against a host-derived viral membrane and the membranes of both Gram-positive and Gram-negative bacteria suggests that the overall helical content in solution under physiological conditions is an important feature for consideration in the design and development of candidate peptide-based antimicrobial compounds.

Handedness Preferences of Heterochiral Helical Peptides Containing Homochiral Peptide Segments

AbstractA homochiral L‐Leu‐L‐Leu‐Aib segment was incorporated into the N‐ or C‐termini of left‐handed (M) helical peptides (D‐Leu‐L‐Leu‐Aib)n. We then investigated the preferred conformations of two sets of three peptides; i.e., Boc‐L‐Leu‐L‐Leu‐Aib‐(D‐Leu‐L‐Leu‐Aib)n‐OMe (n = 1: 1; 2: 2; 3: 3) and Boc‐(D‐Leu‐L‐Leu‐Aib)n‐L‐Leu‐L‐Leu‐Aib‐OMe (n = 1: 4; 2: 5; 3: 6), in solution and in the crystalline state. Nonapeptide 2 and dodecapeptide 3, each containing an N‐terminal L‐Leu‐L‐Leu‐Aib segment, formed left‐handed (M) helices as the preferred secondary structures in solution. In the crystalline state, nonapeptide 2 folded into an (M) α‐helical structure. Peptides 4–6, each containing a C‐terminal L‐Leu‐L‐Leu‐Aib segment, formed roughly equivalent amounts of right‐handed (P) and (M) helices.

Conformation-specific spectroscopy of capped, gas-phase Aib oligomers: tests of the Aib residue as a 310-helix former.

The conformational preferences of a series of capped peptides containing the helicogenic amino acid aminoisobutyric acid (Aib) (Z-Aib-OH, Z-(Aib)2-OMe, and Z-(Aib)4-OMe) are studied in the gas phase under expansion-cooled conditions. Aib oligomers are known to form 310-helical secondary structures in solution and in the solid phase. However, in the gas phase, accumulation of a macrodipole as the helix grows could inhibit helix stabilization. Implementing single-conformation IR spectroscopy in the NH stretch region, Z-Aib-OH and Z-(Aib)2-OMe are both observed to have minor conformations that exhibit dihedral angles consistent with the 310-helical portion of the Ramachandran map (ϕ, ψ = -57°, -30°), even though they lack sufficient backbone length to form 10-membered rings which are a hallmark of the developed 310-helix. For Z-(Aib)4-OMe three conformers are observed in the gas phase. Single-conformation infrared spectroscopy in both the NH stretch (Amide A) and C[double bond, length as m-dash]O stretch (Amide I) regions identifies the main conformer as an incipient 310-helix, having two free NH groups and two C10 H-bonded NH groups, labeled an F-F-10-10 structure, with a calculated dipole moment of 13.7 D. A second minor conformer has an infrared spectrum characteristic of an F-F-10-7 structure in which the third and fourth Aib residues have ϕ, ψ = 75°, -74° and -52°, 143°, Ramachandran angles which fall outside of the typical range for 310-helices, and a dipole moment that shrinks to 5.4 D. These results show Aib to be a 310-helix former in the gas phase at the earliest stages of oligomer growth.

Use of pressure in reversed-phase liquid chromatography to study protein conformational changes by differential deuterium exchange.

The market of protein therapeutics is exploding, and characterization methods for proteins are being further developed to understand and explore conformational structures with regards to function and activity. There are several spectroscopic techniques that allow for analyzing protein secondary structure in solution. However, a majority of these techniques need to use purified protein, concentrated enough in the solution to produce a relevant spectrum. In this study, we describe a novel approach which uses ultrahigh pressure liquid chromatography (UHPLC) coupled with mass-spectrometry (MS) to explore compressibility of the secondary structure of proteins under increasing pressure detected by hydrogen-deuterium exchange (HDX). Several model proteins were used for these studies. The studies were conducted with UHPLC in isocratic mode at constant flow rate and temperature. The pressure was modified by a backpressure regulator up to about 1200 bar. It was found that the increase of retention factors upon pressure increase, at constant flow rate and temperature, was based on reduction of the proteins' molecular molar volume. The change in the proteins' molecular molar volume was caused by changes in protein folding, as was revealed by differential deuterium exchange. The degree of protein folding under certain UHPLC conditions can be controlled by pressure, at constant temperature and flow rate. By modifying pressure during UHPLC separation, it was possible to achieve changes in protein folding, which were manifested as changes in the number of labile protons exchanged to deuterons, or vice versa. Moreover, it was demonstrated with bovine insulin that a small difference in the number of protons exchanged to deuterons (based on protein folding under pressure) could be observed between batches obtained from different sources. The use of HDX during UHPLC separation allowed one to examine protein folding by pressure at constant flow rate and temperature in a mixture of sample solution with minimal amounts of sample used for analysis.

The effect of glycosylation on the antibody recognition of a MUC2 mucin epitope.

MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were characterized in competitive ELISA experiments, and their solution secondary structure was studied by circular dichroism spectroscopy in water and in the ordered structure promoting trifluoroethanol. Our results show that glycosylation in position 19 (peptide (16) PTPT(GalNAcα)GTQ(22) ) resulted in enhanced antibody recognition and significantly altered secondary structure, while glycosylation in position 21 completely demolished the binding. These findings could be useful in determining the nature of antigen-antibody interaction, and perhaps designing synthetic peptide vaccines for tumor therapy.

Structural similarity between β3‐peptides synthesized from β3‐homo‐amino acids and aspartic acid monomers

Formation of stable secondary structures by oligomers that mimic natural peptides is a key asset for enhanced biological response. Here we show that oligomeric β(3)-hexapeptides synthesized from L-aspartic acid monomers (β(3)-peptides 1, 5a, and 6) or homologated β(3)-amino acids (β(3)-peptide 2), fold into similar stable 14-helical secondary structures in solution, except that the former form right-handed 14-helix and the later form left-handed 14-helix. β(3)-Peptides from L-Asp monomers contain an additional amide bond in the side chains that provides opportunities for more hydrogen bonding. However, based on the NMR solution structures, we found that β(3)-peptide from L-Asp monomers (1) and from homologated amino acids (2) form similar structures with no additional side-chain interactions. These results suggest that the β(3)-peptides derived from L-Asp are promising peptide-mimetics that can be readily synthesized using L-Asp monomers as well as the right-handed 14-helical conformation of these β(3)-peptides (such as 1 and 6) may prove beneficial in the design of mimics for right-handed α-helix of α-peptides.

Comparison of the influence of humidity and D-mannitol on the organization of tetraethylene glycol-terminated self-assembled monolayers and immobilized antimicrobial peptides.

We report the use of polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) to characterize the effects of relative humidity (RH) and d-mannitol on the conformations of tetraethylene glycol (EG4)-terminated self-assembled monolayers (SAMs) and immobilized antimicrobial peptides (Cecropin P1 and a hybrid of Cecropin A (1-8) and Melittin (1-18)). These results are used to assess the extent to which d-mannitol can substitute for water in promoting conformational states of the SAMs and oligopeptides similar to those induced by hydration. Our measurements reveal a red shift of the COC asymmetric stretching vibration of the EG4-terminated SAMs with increasing humidity, consistent with a transition from a mixed all-trans/helical (7/2 helix) conformation at 0% RH to a predominantly helical conformation at 90% RH. Significantly, under dry conditions, a thin (2 nm in thickness) overlayer of d-mannitol generated the COC spectroscopic signature of the EG4-terminated SAM measured at high humidity. Comparisons of the effects of humidity and d-mannitol on the secondary structure of the two oligopeptides also revealed both to cause the amide I peak positions, which were measured in dry air (and without d-mannitol) to correspond to α-helical conformations, to undergo red-shifts. The magnitudes of the red-shifts, however, were more pronounced for dry d-mannitol than for high RH, with Cecropin P1 and the hybrid peptide exhibiting amide I peak positions under d-mannitol consistent with bulk aqueous solution secondary structures (random and β-sheet, respectively). These results are discussed in the context of prior reports of the tendency of d-mannitol to form glassy states in the absence of water. Overall, the results presented in this paper support the hypothesis that d-mannitol can substitute, in at least some ways, for the influence of water on the conformational states of biologically relevant molecules at interfaces. The results provide guidance for the design of interfaces for water-free biologics.

γδ T cells recognize the insulin B:9–23 peptide antigen when it is dimerized through thiol oxidation

The insulin peptide B:9–23 is a natural antigen in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). In addition to αβ T cells and B cells, γδ T cells recognize the peptide and infiltrate the pancreatic islets where the peptide is produced within β cells. The peptide contains a cysteine in position 19 (Cys19), which is required for the γδ but not the αβ T cell response, and a tyrosine in position 16 (Tyr16), which is required for both. A peptide-specific mAb, tested along with the T cells, required neither of the two amino acids to bind the B:9–23 peptide. We found that γδ T cells require Cys19 because they recognize the peptide antigen in an oxidized state, in which the Cys19 thiols of two peptide molecules form a disulfide bond, creating a soluble homo-dimer. In contrast, αβ T cells recognize the peptide antigen as a reduced monomer, in complex with the MHCII molecule I-Ag7. Unlike the unstructured monomeric B:9–23 peptide, the γδ-stimulatory homo-dimer adopts a distinct secondary structure in solution, which differs from the secondary structure of the corresponding portion of the native insulin molecule. Tyr16 is required for this adopted structure of the dimerized insulin peptide as well as for the γδ response to it. This observation is consistent with the notion that γδ T cell recognition depends on the secondary structure of the dimerized insulin B:9–23 antigen.

Structural Insights on the Statherin N-Terminal Binding Domain in the Adsorbed State

Hard tissues in living organisms form through protein-mediated mechanisms. Although work is being done to deduce many biomineralization processes, a comprehensive understanding of the organic-inorganic interface remains elusive. One reason for this is the difficulty involved in resolving detailed images of biomolecules in the adsorbed state, as opposed to in solution or as crystals. Surface analysis techniques are excellent at measuring general higher-order structural content, in addition to specific geometric features, but still lack the ability to image whole proteins with atomic precision.A unique partnership emerges from this conundrum. Computer simulation - specifically Molecular Dynamics (MD) - can be used in concert with experiments to provide a more detailed representation of the biomolecule-surface interface. Here we use MD, in conjunction with Parallel Tempering and Metadynamics, to model the first fifteen N-terminal residues of the salivary protein statherin (SN15) in the presence of hydroxyapatite. The SN15 domain lacks any persistent secondary structure in solution, whereas it adopts an α-helical conformation in the adsorbed state. From a converged simulation we show examples of low free energy structures found in the adsorbed state, and compare our own calculated observables to experimental values reported in the literature.While it is true that other researchers have performed detailed structure prediction calculations on a similar system, this work represents an important step for MD-based biomolecule adsorption simulations. Generally, the two approaches can be thought of in terms of the former being a “top-down” method and the latter a “bottom-up” method. Top-down structure prediction sets a certain number of constraints, and then minimizes a system until it arrives at a best fit. Our bottom-up technique builds a statistical ensemble over time, which ultimately yields a converged dataset that can be understood thermodynamically.

Assessing the Structural Dynamics and Membrane Interactions of the Disordered Protein Alpha-Synuclein

Alpha-Synuclein (aSyn) is a small (140-amino acid), soluble neuronal protein that plays a critical role in Parkinson's Disease (PD). Both its native function and its contribution to the disease state are still incompletely understood at the molecular level. aSyn is intrinsically disordered, displaying little or no secondary structure in solution, although it forms an N-terminal (residues 9 to 93) amphipathic α-helix upon binding to lipid bilayers. Moreover, aSyn can act as a membrane curvature-sensing protein and can remodel lipid membranes to induce high curvature. Here we describe our efforts to understand membrane curvature sensing and generation by aSyn. Using single molecule fluorescence and other techniques, we find that while curvature sensing is generally conserved throughout the membrane binding region of aSyn, curvature generation requires specific binding domains. These results will help to form a model for the interplay between aSyn membrane binding activity and membrane remodeling and may have implications for understanding both aSyn's native role as well as its contribution to PD.

Influence of Gold Nanoparticles on the Kinetics of Alpha-Synuclein Aggregation

Alpha-synuclein (AS) is a presynaptic protein lacking a unique secondary structure in solution. AS amyloid aggregates in dopaminergic neurons are the hallmark of Parkinson's disease (PD). The aggregation involves structural transitions from monomeric AS to oligomeric presumably neurotoxic and fibril formation. In spite of its importance for the understanding PD pathobiology and devising rational, targeted therapeutic strategies, details on the aggregation process remain largely unknown. Methodologies and reagents capable of controlling aggregation kinetics are essential tools for the investigation of the molecular amyloid mechanisms. In this work we investigated the influence of citrate capped gold nanoparticles on the aggregation kinetics of AS using a fluorescent probe (MFC) sensitive to the polarity of the molecular microenvironment via an excited state intramolecular proton transfer (ESIPT). The particular effects on the half time, nucleation time and growth rate were ascertained. Gold nanoparticles produced a strong acceleration, with an influence on the nucleation and growth phases of the mechanism. The effects were dependent on the size and concentration of the nanoparticles, being strongest for nanoparticles 10 nm in diameter, with a 3-fold increase in the overall aggregation rate at low concentrations as 20 nM.

Effects of salt concentrations on the structural transitions of peptide-amphiphile solution

ABSTRACTA new peptide amphiphile (PA) called C16-W3K has hierarchical structures, presenting unique solution states, micelle structures, and secondary structures. In this work, the effects of salt (sodium dihydrogenorthophosphate) concentration on the hierarchical structural transitions of the C16-W3K solution due to its active hydrogen bonding in the peptide were discussed. In order to analyze the effects of salt on the structural transitions, the mechanical and structural analyses were conducted by viscosity measurements, transmission electron microscopy (TEM), and circular dichroic (CD) spectroscopy. It was found that the C16-W3K solutions with different salt concentrations presented different multi-scale structural transitions from spherical micelles with α-helix molecular conformations in the sol state to wormlike micelles with β-sheet conformations in the gel state. Additionally, we found that the speed of transition increased as the salt concentration increased and the conformational ratio of β-sheet to α-helix in the solutions increased with the increase in the salt concentration.

RipD (Rv1566c) from Mycobacterium tuberculosis: adaptation of an NlpC/p60 domain to a non-catalytic peptidoglycan-binding function

Enzymes carrying NlpC/p60 domains, for instance RipA and RipB from Mycobacterium tuberculosis, are bacterial peptidoglycan hydrolases that cleave the peptide stems and contribute to cell wall remodelling during cell division. A member of this protein family, RipD (Rv1566c) from M. tuberculosis described in the present study, displays sequence alterations in the NlpC/p60 catalytic triad and carries a pentapeptide repeat at its C-terminus. Bioinformatics analysis revealed RipD-like proteins in eleven mycobacterial genomes, whereas similar pentapeptide repeats occur in cell-wall-localized bacterial proteins and in a mycobacteriophage. In contrast with previously known members of the NlpC/p60 family, RipD does not show peptidoglycan hydrolase activity, which is consistent with the sequence alterations at the catalytic site. A strong interaction of the catalytically inactive core domain with peptidoglycan is however retained, presenting the first example of the NlpC/p60 domains that evolved to a non-catalytic peptidoglycan-binding function. Full-length RipD carrying the C-terminal repeat shows, however, a decrease in binding affinity to peptidoglycan, suggesting that the C-terminal tail modulates the interaction with bacterial cell wall components. The pentapeptide repeat at the C-terminus does not adopt a defined secondary structure in solution which is in accordance with results from the 1.17 Å (1 Å=0.1 nm) crystal structure of the protein carrying two repeat units.

Differentiation and Distributions of DNA/Cisplatin Crosslinks by Liquid Chromatography-Electrospray Ionization-Infrared Multiphoton Dissociation Mass Spectrometry

Liquid chromatography-electrospray ionization-infrared multiphoton dissociation (IRMPD) mass spectrometry was developed to investigate the distributions of intrastrand crosslinks formed between cisplatin and two oligodeoxynucleotides (ODNs), d(A1T2G3G4G5T6A7C8C9C10A11T12) (G3-D) and its analog d(A1T2G3G4G5T6T7C8C9C10A11T12) (G3-H), which have been reported to adopt different secondary structures in solution. Based on the formation of site-specific fragment ions upon IRMPD, two isobaric crosslink products were differentiated for each ODN. The preferential formation of G3G4 and G4G5 crosslinks was determined as a function of reaction conditions, including incubation temperature and presence of metal ions. G3-D consistently exhibited a greater preference for formation of the G4G5 crosslink compared with the G3-H ODN. The ratio of G3G4:G4G5 crosslinks increased for both G3-D and G3-H at higher incubation temperatures or when metal salts were added. Comparison of the IRMPD fragmentation patterns of the unmodified ODNs and the intramolecular platinated crosslinks indicated that backbone cleavage was significantly suppressed near the crosslink.

Effect of pressure on secondary structure of proteins under ultra high pressure liquid chromatographic conditions

There are several spectroscopic techniques such as IR and CD, that allow for analyzing protein secondary structure in solution. However, a majority of these techniques require using purified protein, concentrated enough in the solution, to produce a relevant spectrum. Fundamental principles for the usage of reversed-phase ultra high pressure liquid chromatography (UHPLC) as an alternative technique to study protein secondary structures in solution were investigated. Several “model” proteins, as well as several small ionizable and neutral molecules, were used for these studies. The studies were conducted with UHPLC in isocratic mode, using premixed mobile phases at constant flow rate and temperature. The pressure was modified by a backpressure regulator from about 6000psi to about 12,000psi. It was found that when using a mobile phase composition at which proteins were fully denatured (loss of alpha-helix secondary structure), the retention factors of the proteins increased upon pressure increase in the same manner as non-proteins. When using a mobile phase composition in which proteins were not fully denatured, it was observed that the retention factors of the proteins displayed a much steeper (by one order of magnitude) increase in retention upon pressure increase. It was concluded that in a mobile phase in which the protein is not initially fully denatured, the increase of pressure may facilitate the folding back of the protein to its native state (alpha-helix secondary structure). The impact of different mobile phase compositions on the denaturation of the proteins was studied using CD (Circular Dichroism). Moreover, the effect of flow rate on retention of proteins and small molecules was studied at constant pressure on the different pore size silicas and the impact of internal frictional heating was evaluated.

Charge-neutral morpholino microarrays for nucleic acid analysis

A principal challenge in microarray experiments is to facilitate hybridization between probe strands on the array with complementary target strands from solution while suppressing any competing interactions that the probes and targets may experience. Synthetic DNA analogs, whose hybridization to targets can exhibit qualitatively different dependence on experimental conditions than for nucleic acid probes, open up an attractive alternative for improving selectivity of array hybridization. Morpholinos (MOs), a class of uncharged DNA analogs, are investigated as microarray probes instead of DNA. MO microarrays were fabricated by contact printing of amino-modified probes onto aldehyde slides. In addition to covalent immobilization, MOs were found to efficiently immobilize through physical adsorption; such physically adsorbed probes could be removed by post-printing washes with surfactant solutions. Hybridization of double-stranded DNA targets to MO microarrays revealed a hybridization maximum at intermediate ionic strengths. The decline in hybridization at lower ionic strengths was attributed to an electrostatic barrier accumulated from hybridized DNA targets, whereas at higher ionic strengths it was attributed to stabilization of target secondary structure in solution. These trends, which illustrate ionic strength tuning of forming on-array relative to solution secondary structure, were supported by a stability analysis of MO/DNA and DNA/DNA duplexes in solution.