Study Of Proteins In Solution Research Articles

Quantitative Interpretation of Protein Diffusion Coefficients in Mixed Protiated-Deuteriated Aqueous Solvents.

Diffusion-ordered nuclear magnetic resonance (NMR) spectroscopy is widely used for the analysis of mixtures, dispersing the signals of different species in a two-dimensional spectrum according to their diffusion coefficients. However, interpretation of these diffusion coefficients is typically purely qualitative, for example, to deduce which species are bigger or smaller. In studies of proteins in solution, important questions concern the molecular weight of the proteins, the presence or absence of aggregation, and the degree of folding. The Stokes–Einstein Gierer–Wirtz estimation (SEGWE) method has been previously developed to simplify the complex relationship between diffusion coefficient and molecular mass, allowing the prediction of a species’ diffusion coefficient in a pure solvent based on its molecular weight. Here, we show that SEGWE can be extended to successfully predict both peptide and protein diffusion coefficients in mixed protiated–deuteriated water samples and, hence, distinguish effectively between globular and disordered proteins.

Brownian motion, spin diffusion and protein structure determination in solution

This paper presents my recollections on the development of protein structure determination by NMR in solution from 1968 to 1992. The key to success was to identify NMR-accessible parameters that unambiguously determine the spatial arrangement of polypeptide chains. Inspired by work with cyclopeptides, model considerations showed that enforcing short non-bonding interatomic distances imposes «ring closure conditions» on polypeptide chains. Given that distances are scalar parameters, this indicated an avenue for studies of proteins in solution, i.e., under the regime of stochastic rotational and translational motions at frequencies in the nanosecond range (Brownian motion), where sharp pictures could not be obtained by photography-related methods. Later-on, we used distance geometry calculations with sets of inter-atomic distances derived from protein crystal structures to confirm that measurements of short proton—proton distances could provide atomic-resolution structures of globular proteins. During the years 1976–1984 the following four lines of research then led to protein structure determination by NMR in solution. First, the development of NMR experiments enabling the use of the nuclear Overhauser effect (NOE) for measurements of interatomic distances between pairs of hydrogen atoms in proteins. Second, obtaining sequence-specific resonance assignment solved the “phase problem” for protein structure determination by NMR. Third, generating and programming novel distance geometry algorithms enabled the calculation of atomic-resolution protein structures from limited sets of distance constraints measured by NMR. Fourth, the introduction of two-dimensional NMR provided greatly improved spectral resolution of the complex spectra of proteins as well as efficient delineation of scalar and dipole–dipole 1H–1H connectivities, thus making protein structure determination in solution viable and attractive.

Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography

Hydrostatic pressure in range 0.1–1.5GPa is used to modify biological system behaviour mostly in biophysical studies of proteins in solution. Due to specific influence on the system equilibrium high pressure can act as a filter that enables to identify and investigate higher energy protein conformers. The idea of the presented experiments is to examine the behaviour of RNase A molecule under high pressure before and after introduction of destabilizing mutation. For the first time crystal structures of wild-type bovine pancreatic ribonuclease A and its markedly less stable variant modified at position Ile106 were determined at different pressures. X-ray diffraction experiments at high pressure showed that the secondary structure of RNase A is well preserved even beyond 0.67GPa at room temperature. Detailed structural analysis of ribonuclease A conformation observed under high pressure revealed that pressure influences hydrogen bonds pattern, cavity size and packing of molecule.

A Tabu Search Approach for the NMR Protein Structure-Based Assignment Problem

Spectroscopy is an experimental technique which exploits the magnetic properties of specific nuclei and enables the study of proteins in solution. The key bottleneck of NMR studies is to map the NMR peaks to corresponding nuclei, also known as the assignment problem. Structure-Based Assignment (SBA) is an approach to solve this computationally challenging problem by using prior information about the protein obtained from a homologous structure. NVR-BIP used the Nuclear Vector Replacement (NVR) framework to model SBA as a binary integer programming problem. In this paper, we prove that this problem is NP-hard and propose a tabu search (TS) algorithm (NVR-TS) equipped with a guided perturbation mechanism to efficiently solve it. NVR-TS uses a quadratic penalty relaxation of NVR-BIP where the violations in the Nuclear Overhauser Effect constraints are penalized in the objective function. Experimental results indicate that our algorithm finds the optimal solution on NVRBIP’s data set which consists of seven proteins with 25 templates (31 to 126 residues). Furthermore, it achieves relatively high assignment accuracies on two additional large proteins, MBP and EIN (348 and 243 residues, respectively), which NVR-BIP failed to solve. The executable and the input files are available for download at http://people.sabanciuniv.edu/catay/NVR-TS/NVR-TS.html.

Micro-Raman Spectroscopy of the Hemoglobin Iron-Histidine Bond in Single Erythrocytes

The iron-histidine vibrational mode probes the crucial iron-protein linkage in heme proteins, and it has been established as a sensitive structural probe of the proximal heme pocket from studies of proteins in solution. The cellular environment can modify the interactions and dynamics from the isolated protein. We present Raman measurements on individual erythrocytes under physiological conditions over the frequency range from 150 to 1700 cm-1. Raman spectra were excited with the 442 nm line from a He-Cd laser using 2-3 mW power at the sample. Photodissociation of the ligated hemoglobin is achieved by the excitation laser beam and monitored by the oxidation state marker bands. We investigate the pressure dependence of the iron-histidine stretching mode and compare with results on isolated hemoglobin and myoglobin. A pressure dependent shift suggests a conformational change of the heme environment with pressure.

Using X-rays to Watch Proteins Function with 100 Picosecond Time Resolution

To generate a deeper understanding into the relations between protein structure, dynamics, and function, we have developed time-resolved X-ray methods capable of probing changes in protein structure on time scales as short as ∼100 ps. This infrastructure was first developed on the ID09B time-resolved beamline at the ESRF, and more recently at the ID14B BioCARS beamline at the APS. In these studies, a picosecond laser pulse first photoexcites a protein and triggers a structural change, then a suitably delayed picosecond X-ray pulse passes through the protein and the scattered X-rays are imaged on a 2D detector. When the sample is a protein crystal, this “pump-probe” approach recovers time-resolved diffraction “snapshots” whose corresponding electron density maps can be stitched together into movies that unveil the correlated motions that arise from the photoexcitation event. When the sample is a protein solution, we recover time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) patterns that are sensitive to changes in the size, shape, and structure of the protein. The time-resolved x-ray scattering diffractometer developed at the APS is capable of simultaneously probing both SAXS and WAXS regions with 100 ps time resolution. Importantly, scattering studies of proteins in solution unveil structural dynamics without the constraints imposed by crystal contacts; thus, these scattering “fingerprints” complement results obtained from diffraction studies. Together, these studies provide stringent constraints for putative models of conformational states and structural transitions between them. Time-resolved studies of heme proteins have unveiled at an unprecedented level of structural detail protein conformational dynamics as well as ligand migration and escape. This research was supported in part by the Intramural Research Program of the NIH, NIDDK. JSO is supported by NIH grants GM035469 and HL047020 and Grant C0612 from the Robert Welch A. Foundation.

Small Angle X-Ray Scattering: A Powerful Tool to Analyze Protein Conformation in Solution

In the field of molecular biology and biochemistry in which structural genomics comes as a complement to genome sequencing, Small-Angle X-ray Scattering (SAXS) is an experimental technique that, though not widely known and applied, represents a very powerful tool in the framework of post-genome structural studies. The aim of this review is to present a synthetic description of the basic principles of the theory of SAXS and to discuss in more detail its applications to the study of different biological molecules, focusing the attention to the recent advances in the structural analysis of proteins. These studies are presently undergoing a spectacular expansion associated with the development of powerful data analysis software, with the improvement of the quality of data recorded with synchrotron radiation, and finally with the increasing availability of high resolution three-dimensional structures which can constitute a starting point for the analysis of protein conformations in solution. The advantage of SAXS with respect to other techniques in the structural studies of proteins resides in the possibility of performing the measurements in any desired solvent and in the ability to follow changes of the protein structure which may occur as a response to a variety of stimuli: pH or temperature changes, interaction with small ligands, influence of substrate analogues, chemical or genetic modifications, etc. In this review we describe the principles of SAXS and the most recent methods for data analysis in the field of structural biology and, finally, we report some examples of the use of this technique as a powerful tool to the structural study of proteins in solution. Keywords: electromagnetic wave, zero-angle scattering intensity, Debye Method, ab initio shape, Allosteric Transitions, Multidomain protein, DAM Model

Hydration Study of Proteins in Solution by Microwave Dielectric Analysis

We propose a method for evaluating protein hydration which enables one to evaluate the amount of restrained water and distinguish between “weakly” and “strongly” restrained water on proteins in an aqueous solution using microwave dielectric measurement. Measurements were taken with a precision microwave network analyzer and a thermostated glass cell at 20.0 ± 0.01 °C with an open-end flat-surface coaxial probe. Examined proteins were cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and hemoglobin. The present method is based on the Wagner equation and the Hanai equation for emulsion analysis and assumes that there is “weakly” restrained water on proteins which has a simple Debye-type relaxation in the gigahertz region and that the dielectric constants of hydrated solutes at high-frequency limits are given by the electron polarizations of atom groups. This analysis enabled us to characterize more precisely the hydration state of proteins in water.

A structural study of peptides and proteins containing l-alanine residues by 13C NMR spectroscopy combined with ab initio chemical shift calculations

From the observation of solid-state 13C NMR chemical shifts of l-alanine residues which are contained in some peptides, and the 13C chemical shift calculations employing the coupled-Hartree—Fock method with gauge-invariant-atomic-orbital, it was found that the 13C chemical shift of the C β-carbon in the l-alanine residue is related to the main-chain dihedral angles, φψ, but that of the C α-carbon is affected not only by dihedral angles but also by hodrogen-bonding structure. These results were successfully applied to the structural study of proteins in solution, ribonuclease H from Escherichia coli and basic pancreatic trypsin inhibitor.

Toward automated determination of buildup rates of nuclear overhauser effects in proteins, using symmetry projection operators

The nuclear Overhauser effect is the most widely used spectral NMR parameter in the study of proteins in solution (I, 2). In particular the NOE has been used to determine the three-dimensional structure of proteins in solution ( I, 3-5). However, the NOE is equally important for the study of the dynamical properties of proteins (6) and of protein folding ( 7). In multispin systems it is the cross-relaxation rate which relates to the distance between two protons or to the rate of a chemical exchange event. In the determination of protein structures, however, the cross-relaxation rate has so far rarely been used, and instead the size of a NOE, recorded at a particular mixing time, is used to estimate a distance range for the pair of protons considered. For many purposes this approach has been extremely productive, although it is widely accepted that the use of cross-relaxation rates determined from a series of NOE experiments is the most accurate basis for the determination of distances between protons (8-10). In proteins the accurate distance determination can be hampered by the uncertainties of the contribution of molecular motion to the cross-relaxation rates. This has been one major reason for using just the size of NOES to estimate distance ranges; however, another major reason is the vast effort required to measure the NOE buildup rates for hundreds of NOES in a series of NOE time-development spectra of a protein. Such an effort only becomes feasible if the process can be automated and a reliable numerical integration procedure can be established. Here we present such an approach to automating the analysis of a series of NOE spectra of proteins. The automation is partly based on the use of symmetry projection, which we have previously applied to automated identification of cross peaks of COSY spectra as well as to measurements of coupling constants in E.COSY spectra ( 11,12), The procedure has been applied to a series of two-dimensional NOE spectra of the protein barley serine proteinase inhibitor 2( BSPI-2)) which is one of the few proteins for which both a crystal structure ( 13) and a solution structure is known (4, 5, 14, 15), and the results of this application will be described. In the present work the NOE intensity has been determined using numerical integration. However, several other methods for NOE intensity determination have been proposed. Denk et al. (16) have suggested approximating each experimental NOE

Protein Structure Determination in Solution by Nuclear Magnetic Resonance Spectroscopy

Knowledge of three-dimensional protein structures is one of the foundations of protein design and protein engineering. Nuclear magnetic resonance spectroscopy was recently introduced as a second method for protein structure determination, in addition to the well-established diffraction techniques with protein single crystals. This new approach enables one to carry out detailed structural studies of proteins in solution and other noncrystalline states, which may be similar or identical to the physiological environment, and promises new insights into the dynamics of protein molecules and the protein-folding problem.

One- and two- dimensional 15N/ 1H NMR of filamentous phage coat proteins in solution

High resolution 15N NMR studies of proteins in solution can be performed efficiently by combining the use of isotopically enriched proteins and pulse sequences that generate polarization transfer from protons and result in two-dimensional heteronuclear chemical shift correlation spectra. The coat proteins of the filamentous bacteriophages fd and Pf1 solubilized in detergent micelles give one- and two- dimensional NMR spectra with resolved resonances for nearly all of the nitrogen sites in the proteins. The resonances from the amide sites with slowly exchanging protons can be obtained as a subset of the resonances of all amide sites by comparing the spectra of proteins in D2O and H2O solutions at pH = 4.0.

Assignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solution.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAssignment of the three methionyl carbonyl carbon resonances in Streptomyces subtilisin inhibitor by a carbon-13 and nitrogen-15 double-labeling technique. A new strategy for structural studies of proteins in solutionMasatsune Kainosho and Takashi TsujiCite this: Biochemistry 1982, 21, 24, 6273–6279Publication Date (Print):November 1, 1982Publication History Published online1 May 2002Published inissue 1 November 1982https://doi.org/10.1021/bi00267a036RIGHTS & PERMISSIONSArticle Views294Altmetric-Citations141LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (903 KB) Get e-Alerts Get e-Alerts

Interresidue distance measurements in proteins Fluorescent energy transfer between tryptophans and a Ru(III)(NH 3) 5-histidine complex in α-lytic protease and lysozyme

The mechanism by which the intrinsic fiuorescence of tryptophan residues in α-lytic protease and lysozyme are quenched by a complex formed between the single histidine residue in each protein and Ru(III)(NH 3) 5 was investigated. The R 0 values for α-lytic protease and lysozyme were 15.5 and 11.8 Å, respectively. Good agreement between the efficiency of energy transfer measured experimentally and that calculated from the X-ray data, assuming the Förster dipole-dipole mechanism, demonstrates that this mechanism is appropriate. The ease with which the ruthenium-labeled enzymes can be synthesized and purified suggests that the Ru(III)(NH 3) 5-His complex may have general utility in structural studies of proteins in solution.

Carbon-13 nuclear magnetic resonance studies of proteins.

Nuclear magnetic resonance (NMR) methods, in principle, are capable of resolving resonances of single atoms in protein molecules. In practice, most of the resonances from hydrogen nuclei (lH NMR) in proteins overlap to produce a broad unresolved envelope (1). Carbon-13 NMR (13C NMR) has an intrinsically higher resolving power than proton NMR by a factor of about 20 because of the greater range of electronic configurations experienced by carbon atoms (2). Consequently, initial results on amino acids were interpreted to indicate that I3C NMR would be a potentially valuable tool in studies of proteins in solution (3). Initially, sensitivity problems made this potential unrealizable. The sensitivity of carbon-13 relative to proton NMR at the same magnetic field strength is 1:62.9. If the fact that I3C has a natural abundance of 1.1 % is taken into account (12C has no resonance condition), this leads to a relative sensitivity of 1:5716. This sensitivity problem in l3C NMR, however, has to a large extent been overcome by the introduc­ tion of the pulse Fourier transform method (4). Further losses of signal intensity in BC NMR spectra arise as a result of coupling between 13C and IH nuclear spins. These couplings can be removed by the applica­ tion of a second radiofrequency field at the resonance value for protons. If this frequency has sufficient power and is spread over the entire range of proton absorp­ tions, a process often accomplished by noise modulation, then complete decoupling of the proton nuclear spins from the 13C nuclear spins results. Each carbon atom in the molecule then gives rise to a single resonance, and the resultant proton­ decoupled I3C spectra are relatively simplified. In addition, applying a IH decou­ piing frequency leads to changes in the relative populations of the energy levels for the DC nuclear spins; this phenomenon, known as the nuclear Overhauser effect

Studies of proteins in solution by natural-abundance carbon-13 nuclear magnetic resonance. Spectral resolution and relaxation behavior at high magnetic field strengths.

Studies of proteins in solution by natural-abundance carbon-13 nuclear magnetic resonance. Spectral resolution and relaxation behavior at high magnetic field strengths.

Room temperature phosphorescence and the dynamic aspects of protein structure.

While the phosphorescence of aromatic chromophores in solution is normally quenched through diffusion of dissolved oxygen and other solvent-mediated processes, the phosphorescence of some proteins in solution is observed at room temperature. The tryptophan phosphorescence arises from residues which are hindered from interaction with oxygen by the folding of the polypeptide chains. Measurements of the phosphorescence lifetime of horse liver alcohol dehydrogenase (alcohol: NAD(+) oxidoreductase, EC 1.1.1.1) as a function of oxygen concentration indicate that internal tryptophan residues are periodically exposed to oxygen. This permits the calculation of rate constants for conformational oscillations in the enzyme. The present article illustrates the feasibility of employing phosphorescence in the study of proteins in solution in general and the utility of such experiments in probing the dynamic aspects of protein structure.

Infrared Spectra and Protein Conformations in Aqueous Solutions: II. SURVEY OF GLOBULAR PROTEINS

Infrared spectra in the region of the amide I' band have been determined for a number of proteins in D2O solution. Comparison of the observed band positions with structural analyses based on optical rotatory dispersion and circular dichroism data, as well as with crystallographically determined structures, indicates that this technique may be useful in conformational studies of proteins in solution.

Anaphylaxis to formed or cellular elements

Our present knowledge of anaphylaxis is based almost entirely on the study of proteins in solution, such as blood serum. From the standpoint of infectious disease, the analysis of the immunological reaction to formed elements would appear to be of greater importance. The anaphylactic response to bacteria has regularly been found to be extremely slight. The present report deals with a study of the anaphylactic response to red blood cells.Friedberger reported during the current year that guinea pigs could not be sensitized to alien red blood cells either by the active or by the passive method. On the contrary, these animals can regularly be sensitized by either method provided the proper technique be followed. In order to sensitize actively against alien red blood cells it is essential to give a series (2 or 3) of preliminary injections, instead of the single sensitizing injection which is customary in the case of serum. The reason for this will be obvious from the subsequent data. As regards passive sensit...