RESUMO
Chemical shift perturbation (CSP) is a simple NMR technique for studying the DNA binding of proteins. Titration of the unlabeled DNA into the 15N-labeled protein is monitored by acquiring a two-dimensional (2D) heteronuclear single-quantum correlation (HSQC) spectrum at each step of the titration. CSP can also provide information on the DNA-binding dynamics of proteins, as well as protein-induced conformational changes in DNA. Here, we describe the titration of DNA for the 15N-labeled Z-DNA-binding protein, monitored via 2D HSQC spectra. NMR titration data can be analyzed with the active B-Z transition model to provide the protein-induced B-Z transition dynamics of DNA.
Assuntos
DNA Forma Z , Espectroscopia de Ressonância Magnética/métodos , Imageamento por Ressonância Magnética , Proteínas de Ligação a DNARESUMO
Distal-less 3 (Dlx3) is a homeobox-containing transcription factor and plays a crucial role in the development and differentiation process. Human Dlx3 consists of two transactivation domains and a homeobox domain (HD) that selectively binds to the consensus site (5'-TAATT-3') of the DNA duplex. Here, we performed chemical shift perturbation experiments on Dlx3-HD in a complex with a 10-base-paired (10-bp) DNA duplex under various salt conditions. We also acquired the imino proton spectra of the 10-bp DNA to monitor the changes in base-pair stabilities during titration with Dlx3-HD. Our study demonstrates that Dlx3-HD selectively recognizes its consensus DNA sequences through the α3 helix and L1 loop regions with a unique dynamic feature. The dynamic properties of the binding of Dlx3-HD to its consensus DNA sequence can be modulated by varying the salt concentrations. Our study suggested that this unique structural and dynamic feature of Dlx3-HD plays an important role in target DNA recognition, which might be associated with tricho-dento-osseous syndrome.
Assuntos
Proteínas de Homeodomínio , Sais , Fatores de Transcrição , DNA/metabolismo , Genes Homeobox , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Sais/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune responses, and viral infections. Numerous studies have implicated a role for conformational motions during ZBPs binding upon DNA, but the quantitative intrinsic conformational exchanges of ZBP have not been elucidated. To understand the correlation between the biological function and dynamic feature of the Zα domains of human ADAR1 (hZαADAR1), we have performed the 15N backbone amide Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments on the free hZαADAR1 at two different magnetic fields at 35 °C. The robust inter-dependence of parameters in the global fitting process using multi-magnetic field CPMG profiles allows us characterizing the dynamic properties of conformational changes in hZαADAR1. This study found that free hZαADAR1 exhibited the conformational exchange with a kex of 5784 s-1 between the states "A" (89% population) and "B" (11% population). The different hydrophobic interactions among helices α1, α2, and α3 between these two states might correlate with efficient Z-DNA binding achieved by the hydrogen bonding interactions between its side-chains and the phosphate backbone of Z-DNA.
Assuntos
Adenosina Desaminase/química , Proteínas de Ligação a RNA/química , Adenosina Desaminase/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Edição de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in µs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)-DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1-DNA complex with a slow B-Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s-1, respectively, in agreement with two regimes of residue-dependent chemical shift differences-the "dominant oscillatory regime" and "semi-oscillatory regime". We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.
Assuntos
Adenosina Desaminase/química , DNA de Forma B/química , DNA Forma Z/química , Modelos Moleculares , Proteínas de Ligação a RNA/química , Adenosina Desaminase/metabolismo , Algoritmos , DNA de Forma B/metabolismo , DNA Forma Z/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas de Ligação a RNA/metabolismo , TermodinâmicaRESUMO
Structural transformation of the canonical right-handed helix, B-DNA, to the non-canonical left-handed helix, Z-DNA, can be induced by the Zα domain of the human RNA editing enzyme ADAR1 (hZαADAR1). To characterize the site-specific preferences of binding and structural changes in DNA containing the 2'-O-methyl guanosine derivative (mG), titration of the imino proton spectra and chemical shift perturbations were performed on hZαADAR1 upon binding to Z-DNA. The structural transition between B-Z conformation as the changing ratio between DNA and protein showed a binding affinity of the modified DNA onto the Z-DNA binding protein similar to wild-type DNA or RNA. The chemical shift perturbation results showed that the overall structure and environment of the modified DNA revealed DNA-like properties rather than RNA-like characteristics. Moreover, we found evidence for two distinct regimes, "Z-DNA Sensing" and "Modification Sensing", based on the site-specific chemical shift perturbation between the DNA (or RNA) binding complex and the modified DNA-hZαADAR1 complex. Thus, we propose that modification of the sugar backbone of DNA with 2'-O-methyl guanosine promotes the changes in the surrounding α3 helical structural segment as well as the non-perturbed feature of the ß-hairpin region.
Assuntos
Adenosina Desaminase/química , DNA de Forma B/química , DNA Forma Z/química , Proteínas de Ligação a RNA/química , Adenosina Desaminase/metabolismo , DNA/química , DNA de Forma B/metabolismo , DNA Forma Z/metabolismo , Guanosina/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismoRESUMO
The non-canonical structures of nucleic acids are essential for their diverse functions during various biological processes. These non-canonical structures can undergo conformational exchange among multiple structural states. Data on their dynamics can illustrate conformational transitions that play important roles in folding, stability, and biological function. Here, we discuss several examples of the non-canonical structures of DNA focusing on their dynamic characterization by NMR spectroscopy: (1) G-quadruplex structures and their complexes with target proteins; (2) i-motif structures and their complexes with proteins; (3) triplex structures; (4) left-handed Z-DNAs and their complexes with various Z-DNA binding proteins. This review provides insight into how the dynamic features of non-canonical DNA structures contribute to essential biological processes.
Assuntos
DNA/química , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Animais , DNA/metabolismo , DNA Forma Z/química , DNA Forma Z/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Quadruplex G , Humanos , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Estrutura Molecular , Ácidos Nucleicos/química , Motivos de Nucleotídeos , Ligação ProteicaRESUMO
Liquid-liquid phase separation is common in complex mixtures, but the behavior of nanoconfined liquids is poorly understood from a physical perspective. Dimethyl sulfoxide (DMSO) is an amphiphilic molecule with unique concentration-dependent bulk properties in mixtures with water. Here, we use ultrafast two-dimensional infrared (2D IR) spectroscopy to measure the H-bond dynamics of two probe molecules with different polarities: formamide (FA) and dimethylformamide (DMF). Picosecond H-bond dynamics are fastest in the intermediate concentration regime (20-50 mol % DMSO), because such confined water exhibits bulk-like dynamics. Each vibrational probe experiences a unique microscopic environment as a result of nanoscale phase separation. Molecular dynamics simulations show that the dynamics span multiple time scales, from femtoseconds to nanoseconds. Our studies suggest a previously unknown liquid environment, which we label "local bulk", in which despite the local heterogeneity, the ultrafast H-bond dynamics are similar to bulk water.
RESUMO
Aqueous cosolvent systems (ACoSs) are mixtures of small polar molecules such as amides, alcohols, dimethyl sulfoxide, or ions in water. These liquids have been the focus of fundamental studies due to their complex intermolecular interactions as well as their broad applications in chemistry, medicine, and materials science. ACoSs are fully miscible at the macroscopic level but exhibit nanometer-scale spatial heterogeneity. ACoSs have recently received renewed attention within the chemical physics community as model systems to explore the relationship between intermolecular interactions and microscopic liquid-liquid phase separation. In this perspective, we provide an overview of ACoS spatial segregation, dynamic heterogeneity, and multiscale relaxation dynamics. We describe emerging approaches to characterize liquid microstructure, H-bond networks, and dynamics using modern experimental tools combined with molecular dynamics simulations and network-based analysis techniques.
RESUMO
Dimethyl sulfoxide (DMSO) water mixtures have been widely studied due to their unique concentration-dependent bulk properties. Here, we present an empirical spectroscopic map for the sulfinyl (S=O) stretching mode. The model can be used to interpret infrared (IR) absorption and ultrafast two-dimensional infrared (2D IR) spectra and quantify hydrogen bond populations and lifetimes by directly connecting spectroscopic measurements with structures and dynamics from molecular dynamics simulations. The electrostatic map is directly parameterized against experimental absorption spectra in the S=O stretching region (980-1100 cm-1) of dilute DMSO in water. A comparison of center peak frequencies shows that the map performs well across the entire DMSO concentration range, accurately reproducing the â¼10 cm-1 red-shift per hydrogen bond observed in the experiments. We further benchmark the map by comparing experimental and simulated 2D IR spectra generated by direct numerical integration of the Schrödinger equation. We expect that this empirical frequency map will provide a quantitative platform for investigating intermolecular interactions, microscopic heterogeneity, and ultrafast dynamics in complex liquid mixtures containing DMSO.
RESUMO
Up to 40% of intracellular water is confined due to the dense packing of macromolecules, ions, and osmolytes. Despite the large body of work concerning the effect of additives on the biomolecular structure and stability, the role of crowding and heterogeneity in these interactions is not well understood. Here, infrared spectroscopy and molecular dynamics simulations are used to describe the mechanisms by which crowding modulates hydrogen bonding interactions between water and dimethyl sulfoxide (DMSO). Specifically, we use formamide and dimethylformamide (DMF) as molecular crowders and show that the SâO hydrogen bond populations in aqueous mixtures are increased by both amides. These additives increase the amount of water within the DMSO first solvation shell through two mechanisms: (a) directly stabilizing water-DMSO hydrogen bonds; (b) increasing water exposure by destabilizing DMSO-DMSO self-interactions. Further, we quantified the hydrogen bond enthalpies between the different components: DMSO-water (61 kJ/mol) > DMSO-formamide (32 kJ/mol) > water-water (23 kJ/mol) â« formamide-water (4.7 kJ/mol). Spectra of carbonyl stretching vibrations show that DMSO induces the dehydration of amides as a result of strong DMSO-water interactions, which has been suggested as the main mechanism of protein destabilization.
RESUMO
Dimethyl sulfoxide (DMSO) disrupts the hydrogen-bond networks in water. The widespread use of DMSO as a cosolvent, along with its unusual attributes, have inspired numerous studies. Herein, infrared absorption spectroscopy of the S=O stretching mode combined with molecular dynamics and quantum chemistry models were used to directly quantify DMSO/water hydrogen-bond populations in binary mixtures. Singly H-bonded species are dominant at 10â mol %, due to strong DMSO-water interactions. We found an unexpected increase in non-hydrogen-bonded DMSO near the eutectic point (ca. 35â mol %) which also correlates with several abnormalities in the bulk solution properties. We find evidence for three distinct regimes: 1)â strong DMSO-water interactions (<30â mol %), 2)â ideal-solution-like (30-90â mol %), and 3)â self-interaction, or aggregation, regime (>90â mol %). We propose a "step in" mechanism, which involves hydrogen bonding between water and the DMSO aggregate species.
RESUMO
Site-selective isotopic labeling of amide carbonyls offers a nonperturbative means to introduce a localized infrared probe into proteins. Although this strategy has been widely used to investigate various biological questions, the dependence of the underlying amideâ I vibrational frequency on electric fields (or Stark tuning rate) has not been fully determined, which prevents it from being used in a quantitative manner in certain applications. Herein, through the use of experiments and molecular dynamics simulations, the Stark tuning rate of the amideâ I vibration of an isotopically labeled backbone carbonyl in a transmembrane α-helix is determined to be approximately 1.4â cm(-1) /(MV/cm). This result provides a quantitative basis for using this vibrational model to assess local electric fields in proteins, among other applications. For instance, by using this value, we are able to show that the backbone region of a dipeptide has a surprisingly low dielectric constant.
Assuntos
Amidas/química , Eletricidade , Simulação de Dinâmica Molecular , Peptídeos/química , VibraçãoRESUMO
Despite our extensive understanding of water-soluble protein folding kinetics, much less is known about the folding dynamics and mechanisms of membrane proteins. However, recent studies have shown that for relatively simple systems, such as peptides that form a transmembrane α-helix, helical dimer, or helix-turn-helix, it is possible to assess the kinetics of several important steps, including peptide binding to the membrane from aqueous solution, peptide folding on the membrane surface, helix insertion into the membrane, and helix-helix association inside the membrane. Herein, we provide a brief review of these studies and also suggest new initiation and probing methods that could lead to improved temporal and structural resolution in future experiments.
Assuntos
Membrana Celular/química , Membranas Artificiais , Peptídeos/química , Dobramento de Proteína , Animais , Humanos , Cinética , Estrutura Secundária de ProteínaRESUMO
For nuclear magnetic resonance (NMR)-based protein structure determinations, the random coil chemical shifts are very important because the secondary and tertiary protein structure predictions become possible by examining deviations of measured chemical shifts from those reference chemical shift values. In addition, neighboring residue effects on chemical shifts and J-coupling constants are crucial in understanding the nature of conformational propensities exhibited by unfolded or intrinsically disordered proteins. We recently reported the 1D NMR results for a complete set of terminally blocked dipeptides (Oh KI, Jung YS, Hwang GS, Cho M. J Biomol NMR 2012;53:25-41), but the NMR resonance assignments were not possible so that the average chemical shifts and J-coupling constants were only considered. In the present work, to thoroughly investigate the neighboring residue effects and random coil chemical shifts we extend the previous studies with 2D NMR, and measured all the (3) J(HNHα) values and H(α) and H(N) chemical shifts of the same set of terminally blocked dipeptides that are free from structural effects like secondary structure, hydrogen-bond, long-range backbone, and side-chain interactions. In particular, the preceding and following residue effects on amino-acid backbone conformational propensities are revealed and directly compared with previous works on either short peptides or empirical chemical shift database.
Assuntos
Dipeptídeos/química , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Understanding intrinsic conformational preferences of amino-acids in unfolded proteins is important for elucidating the underlying principles of their stability and re-folding on biological timescales. Here, to investigate the neighbor interaction effects on the conformational propensities of amino-acids, we carried out (1)H NMR experiments for a comprehensive set of blocked dipeptides and measured the scalar coupling constants between alpha protons and amide protons as well as their chemical shifts. Detailed inspection of these NMR properties shows that, irrespective of amino-acid side-chain properties, the distributions of the measured coupling constants and chemical shifts of the dipeptides are comparatively narrow, indicating small variances of their conformation distributions. They are further compared with those of blocked amino-acids (Ac-X-NHMe), oligopeptides (Ac-GGXGG-NH(2)), and native (lysozyme), denatured (lysozyme and outer membrane protein X from Escherichia coli), unstructured (Domain 2 of the protein 5A of Hepatitis C virus), and intrinsically disordered (hNlg3cyt: intracellular domain of human NL3) proteins. These comparative investigations suggest that the conformational preferences and local solvation environments of the blocked dipeptides are quite similar to not only those of other short oligopeptides but also those of denatured and natively unfolded proteins.
Assuntos
Dipeptídeos/química , Proteínas/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Dipeptídeos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Hidrolases/química , Hidrolases/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Desnaturação Proteica , Desdobramento de Proteína , Proteínas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Despite prolonged scientific efforts to elucidate the intrinsic peptide backbone preferences of amino-acids based on understanding of intermolecular forces, many open questions remain, particularly concerning neighboring peptide interaction effects on the backbone conformational distribution of short peptides and unfolded proteins. Here, we show that spectroscopic studies of a complete library of 400 dipeptides reveal that, irrespective of side-chain properties, the backbone conformation distribution is narrow and they adopt polyproline II and ß-strand, indicating the importance of backbone peptide solvation and electronic effects. By directly comparing the dipeptide circular dichroism and NMR results with those of unfolded proteins, the comprehensive dipeptides form a complete set of structural motifs of unfolded proteins. We thus anticipate that the present dipeptide library with spectroscopic data can serve as a useful database for understanding the nature of unfolded protein structures and for further refinements of molecular mechanical parameters.
Assuntos
Dipeptídeos/química , Biblioteca de Peptídeos , Desdobramento de Proteína , Aminoácidos/química , Dicroísmo Circular , Dipeptídeos/síntese química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Proteica , Estabilidade Proteica , Solventes/química , Eletricidade Estática , Água/químicaRESUMO
Despite that a number of experimental and theoretical investigations have been carried out to determine the structure of trialanine in water, the reported populations of polyproline II (PPII) and ß-strand conformers vary and were found to be dependent on which spectroscopic method was used. Such discrepancies are due to limitations of different spectroscopic methods used. Here, the temperature- and pH-dependent circular dichroism (CD) and NMR experiments have been carried out to develop a self-consistent singular value decomposition procedure. The temperature-dependent CD spectra indicate the presence of two conformers, but due to the two peptide bonds in a trialanine, one should take into consideration of four different conformers to fully interpret the NMR results. From the pH-dependent NMR coupling constant measurements, the conformation of zwitterionic trialanine is little different from that of cationic one. The strong pH dependency of CD spectrum is likely due to charge transfer transitions between carboxylate and nearby peptide groups or internal field effects not to pH-dependent conformational change. To simultaneously analyze the temperature-dependent CD and NMR data, a self-consistent procedure was used to newly determine the reference NMR coupling constants required to estimate one of the peptide dihedral angles. From the estimated enthalpy and entropy changes associated with the transition from enthalpically favorable PPII conformer to entropically favorable ß-strand conformer, the relative populations of the four possible conformers of trialanine were determined and compared with the previous experimental findings. We anticipate that the present experimental results and interpretation procedure would be of use in determining the solution structures of small oligopeptides in the future.
Assuntos
Oligopeptídeos/química , Peptídeos/química , Água/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
To study the azido gauche effect on the backbone conformation of ß-azidoalanine (Aza) dipeptide (AAD, Ac-Aza-NHMe) and tripeptide (AAT, Ac-Aza-Aza-NH(2)), we used spectroscopic methods in combination with quantum chemistry calculations and molecular dynamics (MD) simulations. From the (1)H NMR coupling constants and (1)H,(1)H NOESY experimental data, we found that AAD in water mainly adopts a seven-membered cyclic (C(7)) rather than polyproline II (P(II)) backbone conformation and prefers the gauche- (g(-)) side-chain conformer. From the amide I IR absorption and circular dichroism (CD) spectra, the backbone conformation of AAD in water is found to deviate from P(II) but is rather close to C(7). Thus, the backbone conformation of AAD differs from that of alanine dipeptide (AD, Ac-Ala-NHMe), which is mainly P(II) in water. The underlying origin of the backbone conformational difference between AAD and AD in water was elucidated by quantum chemistry calculations with density functional theory (DFT). It was found that the C(7)/g(-) conformer is the lowest energy structure of an isolated AAD. Here, the ß-azido group forms intramolecular electrostatic interactions with two neighboring peptide bonds, which are facilitated by the azido gauche effect. Thus, the ß-azido group appears to be responsible for directing the peptide backbone conformation toward the C(7) structure. The quantum mechanical/molecular mechanical (QM/MM) MD simulations show that AAD in water adopts neither P(II) nor right-handed α-helix (α(R)) and prefers the g(-) conformer. Thus, the intramolecular electrostatic interactions between the ß-azido group and two nearby peptide bonds are also found even in the aqueous solution structure of AAD. Consequently, the ß-azido group appears to be an effective C(7)-conformation-directing element, which may also be useful for tuning the structures of other amino acids and polypeptides.
Assuntos
Alanina/análogos & derivados , Azidas/química , Peptídeos/química , Alanina/química , Dicroísmo Circular , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Teoria Quântica , Espectrofotometria Infravermelho , Eletricidade EstáticaRESUMO
A variety of spectroscopic probe molecules have been used to study the local electrostatic environment in proteins. Particularly, a few IR probes such as nitrile- and thiocyanate-derivatized amino acids were found to be quite useful not just because they are small but also because their IR absorption frequencies strongly depend on the strengths of hydrogen bonds with the surrounding protic solvent molecules. Recently, we experimentally demonstrated that azido-derivatized alanine is an excellent IR probe for studying structural change in protein in solution. The asymmetric stretching mode frequency of N(3)-group becomes blueshifted when it is dissolved in water. Such a blueshifting behavior upon hydrogen-bonding interaction with protic solvent molecules was commonly found in other IR probes containing a triple bond such as CN and SCN groups. In this paper, theoretical descriptions on the solvatochromic frequency shift and fluctuation of the azido stretch frequency are presented by carrying out ab initio calculations and both classical and quantum mechanical/molecular mechanical dynamics simulation studies for azidomethane and azidoalanine dipeptide dissolved in water. Two different methods developed here are based on the distributed multipole interaction models, and they are shown to be useful to describe site-specific hydrogen-bonding interaction-induced red- or blueshift of the azido stretch frequency. To test the validity of thus obtained interpolation formula, numerically simulated IR spectra of azidomethane and azidoalanine dipeptide in water are directly compared with experimental results. We anticipate that the present theoretical approaches will be of use in connecting experimentally measured azido stretch frequency to conformational change in protein containing this azido-derivatized alanine residue.
Assuntos
Azidas/química , Raios Infravermelhos , Teoria Quântica , Eletricidade Estática , Absorção , Alanina/química , Dipeptídeos/química , Modelos Químicos , Solventes/química , Vibração , Água/químicaRESUMO
Beta-azidoalanine dipeptide 1 was synthesized, and its azido stretching vibration in H2O and dimethyl sulfoxide (DMSO) was studied by using Fourier transform (FT) IR spectroscopy. The dipole strength of the azido stretch mode is found to be about 19 and 5 times larger than those of the CN and SCN stretch modes, respectively, which have been used as local environmental IR sensors. The azido stretch band in H2O is blue-shifted by about 14 cm(-1) in comparison to that in DMSO, indicative of its sensitivity to the electrostatic environment. To test the utility of beta-azidoalanine as an IR probe of the local electrostatic environment in proteins, azidopeptide 4 was prepared by its incorporation into Abeta(16-22) peptide of the Alzheimer's disease amyloid beta-protein at position Ala21. The amide I IR spectrum of 4 in D2O suggests that the azidopeptide thus modified forms in-register beta-sheets in aggregates as observed for normal Abeta(16-22). The azido peak frequency of 4 in aggregates is almost identical to that in DMSO, indicating that the azido group is not exposed to water but to the hydrophobic environment. We believe that beta-azidoalanine will be used as an effective IR probe for providing site-specific information about the local electrostatic environments of proteins.