Dielectric relaxation in a single tryptophan protein.

Red-edge anisotropy microscopy enables dynamic imaging of homo-FRET between green fluorescent proteins in cells

Abstract: Steady-state fluorescence anisotropy measurements can be used to detect fluorescence resonance energy transfer (FRET) between identical fluorophores (homo-FRET). However, the contribution of homo-FRET to the steady-state anisotropy must be discerned from those due to the orientational distribution and rotational diffusion, which so far has required photobleaching controls, largely precluding dynamic measurements in live cells. We describe a variation of steady-state anisotropy microscopy in which the contribution of homo-FRET is dynamically isolated from the total anisotropy by exploiting the loss of energy transfer that occurs at red-edge excitation. Excitation of enhanced green fluorescent protein (EGFP) at the red-edge of its absorption band shows the shift in the emission spectrum compared to main-band excitation that is characteristic for photo-selection of static low energy S(0)-S(1) transitions that fail to exhibit FRET. An experimental setup for steady-state fluorescent anisotropy microscopy is described that can be used to acquire anisotropy images in live cells at main-band and red-edge excitation of EGFP. We demonstrate in live cells homo-FRET suppression of protein fusion constructs that consist of two and three EGFP molecules connected by short linkers. This methodology represents a novel approach for the dynamic measurement of homo-FRET in live cells that will be of utility in the biological sciences to detect oligomerization and concentration dependent interactions between identically labeled molecules.

Novel insights into protein structure and dynamics utilizing the red edge excitation shift approach

Abstract: A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a corresponding shift in the excitation wavelength toward the red edge of the absorption band, is termed the red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which depends on the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise ‘optically silent’ water molecules. This makes REES extremely useful since hydration plays a crucial modulatory role in the formation and maintenance of organized molecular assemblies such as folded proteins in aqueous solutions and biological membranes. The application of REES as a powerful tool to monitor the organization and dynamics of a variety of soluble, cytoskeletal, and membrane-bound proteins is discussed

Precursor of the Inactive 2S Seed Storage Protein from the Indian Mustard Brassica juncea Is a Novel Trypsin Inhibitor CHARACTERIZATION, POST-TRANSLATIONAL PROCESSING STUDIES, AND TRANSGENIC EXPRESSION TO DEVELOP INSECT-RESISTANT PLANTS

Abstract: A number of trypsin inhibitor (TI) genes have been used to generate insect-resistant plants. Here we report a novel trypsin inhibitor from Indian mustard Brassica juncea (BjTI) that is unique in being the precursor of a 2S seed storage protein. The inhibitory activity is lost upon processing. The predicted amino acid sequence of the precursor based on the B. juncea 2S albumin (Bj2S) gene cloned and sequenced in this laboratory (Bj2Sc; GenBank(TM) accession number ) showed a soybean-TI active site-like motif GPFRI at the expected processing site. The BjTI was found to be a thermostable Kunitz type TI that inhibits trypsin at a molar ratio of 1:1. The 20-kDa BjTI was purified from midmature seeds and found to be processed in vitro to 9- and 4-kDa subunits upon incubation with seed extract. The Bj2Sc sequence was expressed in Escherichia coli pET systems as the inhibitor precursor. The radiolabeled gene product was expressed in vitro in a coupled transcription-translation system and showed the expected processing into subunits. Two in vitro expressed pre-2S proteins, mutated at Gly and Asp residues, were processed normally to mature subunits, showing thereby no absolute requirement of Gly and Asp residues for processing. Finally, the 2S gene was introduced into tobacco and tomato plants. Third generation transgenics expressing BjTI at 0.28-0.83% of soluble leaf proteins showed remarkable resistance against the tobacco cutworm, Spodoptera litura. This novel TI can be used in transforming seed crops for protection to their vegetative parts and early seed stages, when insect damage is maximal; as the seeds mature, the TI will be naturally processed to the inactive storage protein that is safe for consumption.

The Gas-Phase Absorption Spectrum of a Neutral GFP Model Chromophore

Abstract: We have studied the gas-phase absorption properties of the green fluorescent protein (GFP) chromophore in its neutral (protonated) charge state in a heavy-ion storage ring. To accomplish this we synthesized a new molecular chromophore with a charged NH(3) group attached to a neutral model chromophore of GFP. The gas-phase absorption cross section of this chromophore molecule as a function of the wavelength is compared to the well-known absorption profile of GFP. The chromophore has a maximum absorption at 415 +/- 5 nm. When corrected for the presence of the charged group attached to the GFP model chromophore, the unperturbed neutral chromophore is predicted to have an absorption maximum at 399 nm in vacuum. This is very close to the corresponding absorption peak of the protein at 397 nm. Together with previous data obtained with an anionic GFP model chromophore, the present data show that the absorption of GFP is primarily determined by intrinsic chromophore properties. In other words, there is strong experimental evidence that, in terms of absorption, the conditions in the hydrophobic interior of this protein are very close to those in vacuum.

Napin from Brassica juncea: Thermodynamic and structural analysis of stability

Abstract: The napin from Brassica juncea, oriental mustard, is highly thermostable, proteolysis resistant and allergenic in nature. It consists of two subunits – one small (29 amino acid residues) and one large (86 amino acids residues) – held together by disulfide bonds. The thermal unfolding of napin has been followed by differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. The thermal unfolding is characterized by a three state transition with $T_{M1}$ and $T_{M2}$ at 323.5 K and 335.8 K, respectively; $\Delta C_{P1}$ and $\Delta C_{P2}$ are $2.05 kcal mol^{-1} K^{-1}$ and $1.40 kcal mol^{−1} K^{−1}$, respectively. In the temperature range 310–318 K, the molecule undergoes dimerisation. Isothermal equilibrium unfolding by guanidinium hydrochloride also follows a three state transition, N⇆I⇆U with $\Delta G_{1H2O}$ and $\Delta G_{2H2O}$ values of $5.2 kcal mol^{−1}$ and $5.1 kcal mol^{−1}$ at 300 K, respectively. Excess heat capacity values obtained, are similar to those obtained from DSC measurements. There is an increase in hydrodynamic radius from $20 \AA$ to $35.0 \AA$ due to unfolding by guanidinium hydrochloride. In silico alignment of sequences of napin has revealed that the internal repeats (40%) spanning residues 31 to 60 and 73 to 109 are conserved in all Brassica species. The internal repeats may contribute to the greater stability of napin. A thorough understanding of the structure and stability of these proteins is essential before they can be exploited for genetic improvements for nutrition.

Principles of fluorescence spectroscopy

Abstract: Fluorescence methods are being used increasingly in biochemical, medical, and chemical research. This is because of the inherent sensitivity of this technique. and the favorable time scale of the phenomenon of fluorescence. 8 Fluorescence emission occurs about 10- sec (10 nsec) after light absorp tion. During this period of time a wide range of molecular processes can occur, and these can effect the spectral characteristics of the fluorescent compound. This combination of sensitivity and a favorable time scale allows fluorescence methods to be generally useful for studies of proteins and membranes and their interactions with other macromolecules. This book describes the fundamental aspects of fluorescence. and the biochemical applications of this methodology. Each chapter starts with the -theoreticalbasis of each phenomenon of fluorescence, followed by examples which illustrate the use of the phenomenon in the study of biochemical problems. The book contains numerous figures. It is felt that such graphical presentations contribute to pleasurable reading and increased understand ing. Separate chapters are devoted to fluorescence polarization, lifetimes, quenching, energy transfer, solvent effects, and excited state reactions. To enhance the usefulness of this work as a textbook, problems are included which illustrate the concepts described in each chapter. Furthermore, a separate chapter is devoted to the instrumentation used in fluorescence spectroscopy. This chapter will be especially valuable for those perform ing or contemplating fluorescence measurements. Such measurements are easily compromised by failure to consider a number of simple principles."

Mechanisms of Tryptophan Fluorescence Shifts in Proteins

Abstract: Tryptophan fluorescence wavelength is widely used as a tool to monitor changes in proteins and to make inferences regarding local structure and dynamics. We have predicted the fluorescence wavelengths of 19 tryptophans in 16 proteins, starting with crystal structures and using a hybrid quantum mechanical-classical molecular dynamics method with the assumption that only electrostatic interactions of the tryptophan ring electron density with the surrounding protein and solvent affect the transition energy. With only one adjustable parameter, the scaling of the quantum mechanical atomic charges as seen by the protein/solvent environment, the mean absolute deviation between predicted and observed fluorescence maximum wavelength is 6 nm. The modeling of electrostatic interactions, including hydration, in proteins is vital to understanding function and structure, and this study helps to assess the effectiveness of current electrostatic models.

Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies.

Abstract: Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.

Dynamics of Solvation and Charge Transfer Reactions in Dipolar Liquids

Abstract: The effects of solvent and solvent dynamics on chemical reactions, especially on charge transfer processes, have long been a subject of great importance in physical chemistry. In the past, attention was focused pri­ marily on equilibrium solvent effects, such as the effect of solvent polarity on the reaction potential surface. Tn recent years it has become clear that in many fast reactions solvent dynamics can play a direct role and can affect both the rate and the outcome of a reaction profoundly. Thus, an understanding of the time-dependent response of a polar solvent to a changing charge distribution in a polar solute molecule is essential to understand the role of solvent in many important chemical and biological processes in liquids. Such understanding can be achieved by studying the dynamics of solvation of a newly created ion or of an instantaneously changed dipole in a polar liquid. This subject has undergone a renaissance in recent years because of the availability of ultra-short laser pulses that make it possible to study solvation dynamics directly with a time resolution hitherto impossible. An understanding of the details of solvent response to a sudden change in the charge distribution of a polar solute "probe" molecule is beginning to emerge. Experimental studies on the dynamics of solvation are usually carried out by instantaneously creating a charged species inside a polar solvent and subsequently monitoring the emission/absorption spectrum of this

Ultraviolet Spectroscopy of Proteins

Abstract: 1. Spectroscopic Properties of Protein Chromophores.- 1.1 Basic Principles and Definitions of Light Absorption and Emission Spectroscopy.- 1.1.1 Absorption.- 1.1.2 Emission.- 1.2 Light Absorption by the Amide Chromophore..- 1.3 The Absorption Spectra of Amino Acid Residues and Their Analogs.- 1.3.1 Phenylalanine..- 1.3.2 Tyrosine.- 1.3.3 Tryptophan.- 1.3.4 Other Protein Chromophores.- 1.4 Emission Properties of Aromatic Amino Acids.- 1.4.1 Phenylalanine.- 1.4.2 Tyrosine.- 1.4.3 Tryptophan.- 1.5 Conclusions.- 2. Display of Intramolecular and Intermolecular Interactions in Electronic Spectra of Amino Acids and Proteins.- 2.1 Spectroscopic Analysis of the Environmental Polarity and Polarizability Effects.- 2.2 Spectroscopic Manifestation of the Hydrogen Bond.- 2.3 Substitution and Charge Effects.- 2.4 Charge-Transfer Complexes.- 2.5 Broadening of Electronic Spectra.- 2.6 Excited State Processes.- 2.6.1 Solvent Relaxation.- 2.6.2 Exciplexes.- 2.6.3 Excited State Proton Transfer.- 2.6.4 Excited State Electron Transfer.- 2.6.5 On the Nature of the Emitting State of Indole and Tryptophan.- 2.7 Conclusions.- 3. Difference Spectra of Proteins *.- 3.1 Informational Significance of Difference Spectra.- 3.2 Studies of Protein Denaturation.- 3.3 Functional Transformations and Association of Proteins.- 3.3.1 Conformational Changes Under Activation of Precursor Proteins.- 3.3.2 Formation of Enzyme-Substrate and Enzyme-Inhibitor Complexes. Studies on Mechanisms of Catalytic Activity.- 3.3.3 Interaction of Proteins with Small Molecules and Ions.- 3.3.4 Association of Subunits and Formation of Supermolecular Structures.- 3.4 Solvent Perturbation Difference Spectra and Studies in Surface Topography.- of Protein Molecules.- 3.5 Protein-Model Difference Spectra.- 3.6 Conclusions.- 4. Thermal Perturbation Difference Spectroscopy and Temperature-Dependent Conformational Transitions of Proteins.- 4.1 Characteristics of Tyrosine, Tryptophan, and Phenylalanine Spectra and Their Origin.- 4.1.1 Quantitative Analysis of the Experimental Data. Account of Correction Factors.- 4.1.2 TPDS of Tryptophan, Tyrosine, and Phenylalanine.- 4.1.3 Origin of Thermal Perturbation Difference Spectra.- 4.1.4 On the Nature of Longwave Shift with the Temperature Rise.- 4.2 Studies of Thermal Perturbation of Tyrosine, Tryptophan, and Phenylalanine Residues in Proteins.- 4.2.1 Drop in Intensity of TPDS.- 4.2.2 Longwave Shift of TPDS Maxima.- 4.2.3 Absence of Tyrosine Maxima at 287-289 nm.- 4.2.4 Presence of TPDS Maxima in the Region of 300-307 nm.- 4.3 TPDS and Protein Conformational Transitions Depending on Temperature.- and pH of the Medium.- 4.4 Conclusions.- 5. Derivative Spectroscopy of Aromatic Amino Acids and Proteins.- 5.1 The Theoretical Grounds.- 5.2 Derivative Spectra of Tryptophan, Tyrosine, and Phenylalanine.- 5.3 Influence of Solvents on the Derivative Spectra of Aromatic Amino Acids.- 5.4 Analysis of Chromophore Environment in Proteins.- 5.4.1 The State of Phenvlalanine Residues.- 5.4.2 The State of Tyrosine and Tryptophan.- 5.5 Studies of Conformational Transitions in Proteins. Difference-Derivative Spectroscopy.- 5.6 Studies on Broadening of Absorption Spectra.- 5.7 Conclusions.- 6. Spectrophotometric Titration of Proteins.- 6.1 The Spectrophotometric Titration Method.- 6.2 Titration of Tyrosine Residues at Alkaline pH.- 6.3 Conclusions.- 7. Fluorescence Molecular Relaxation Spectroscopy.- 7.1 Relaxational Shift of the Fluorescence Spectra.- 7.2 Time-Resolved Spectroscopy.- 7.3 Edge Excitation Fluorescence Spectroscopy.- 7.3.1 Physical Principles.- 7.3.2 The Effect of Selective Excitation on Fluorescence Spectra of Indole and Tryptophan.- 7.3.3 The Structural Relaxation of Indolic Chromophore in Proteins.- 7.4 Conclusions.- 8. Fluorescence Quenching.- 8.1 Effects of External Difiusional Quenchers.- 8.2 Quenching by Protein Internal Groups and Its Temperature Dependence.- 8.3 Conclusions.- 9. Nonradiative Transfer of Electronic Excitation Energy.- 9.1 General Theory.- 9.2 The Effect of Inhomogeneous Broadening of Spectra and Molecular Relaxation.- on the Nonradiative Transfer of Energy.- 9.3 The Energy Transfer Between Aromatic Amino Acid Residues in Proteins.- 9.4 Conclusions.- 10. Fluorescence Polarization and Rotational Mobility.- 10.1 The Method of Fluorescence Depolarization.- 10.2 The Effect of Dipolar-Reorientational Relaxation on Rotational Depolarization of Fluorescence.- 10.3 Intramolecular Mobility in Proteins as Estimated by Data on Fluorescence Polarization 204 10.4 Conclusions.- 11. Intrinsic Phosphorescence of Proteins.- 11.1 General Mechanisms of Phosphorescence.- 11.1.1 The Principle of the Method,.- 11.1.2 Inhomogeneous Broadening and Molecular Relaxations in Spectroscopy of the Triplet State.- 11.1.3 Indole and Tryptophan Phosphorescence.- 11.2 Phosphorescence Assay of Protein Structures.- 11.2.1 Low-Temperature Phosphorescence.- 11.2.2 Temperature Dependence of Phosphorescence Parameters.- 11.2.3 Phosphorescence of Proteins and Membranes at Room Temperature.- 11.2.4 Phosphorescence and Optically Detected Magnetic Resonance.- 11.3 Conclusions.- 12. Employment of Ultraviolet Spectroscopy in Analytical Chemistry of Proteins.- 12.1 A Method for the Quantitative Spectroscopic Determination of Protein Concentration.- 12.1.1 Determination of Protein Concentration in the 280 nm Region.- 12.1.2 Determination of Protein Concentration in the Far-Ultraviolet Region.- 12.1.3 Determination of Protein Concentration in the Presence of Other Light-Absorbing Substances.- 12.2 Determination of Tryptophan, Tyrosine, and Phenylalanine Content in Proteins.- 12.2.1 Determination of Tyrosine and Tryptophan Concentration by Absorption Spectra..- 12.2.2 Determination of Tyrosine, Tryptophan, and Phenylalanine by Use of Derivatives of the Absorption Spectrum.- 12.2.3 Employment of the Method of Difference and Thermal Perturbation Difference Spectra.- 12.2.4 Fluorescence Spectroscopic Tryptophan and Tyrosine Assay.- 12.3 Spectroscopic Studies of Peptide Bond Splitting in Protein Hydrolysis.- 12.4 Study of the Modification and Oxidation of Chromophore Groups.- 12.5 Hydrogen-Deuterium Exchange in Peptides and Proteins.- 12.6 Conclusions.- 13. Experimental Technique in Protein Spectroscopy.- 13.1 Spectrophotometric Analysis.- 13.1.1 Spectrophotometers, Their Main Characteristics and Calibration.- 13.1.2 Peculiarities of Recording the Difference Spectra of Proteins.- 13.1.3 Difference Spectra of Protein Preparations Differing in Initial Concentration.- 13.1.4 The Recording of Derivative Spectra.- 13.1.5 Turbidity of Protein Preparation and Possibilities of Its Account.- 13.2 Technique of Luminescence Studies.- 13.2.1 Steady State Spectra of Excitation and Fluorescence.- 13.2.2 Technique of Polarization Measurements.- 13.2.3 Technique of Time-Resolved Fluorimetry.- 13.2.4 Technique of Phosphorescence Studies.- 13.3 Multiparametric Detection and Analysis of Spectroscopic Data.- 13.4 Conclusions.- 14. General Conclusions and Prospects.- 14.1 Advantages and Limitations of Molecular Probe Methods.- 14.2 The Comparative Analysis of UV-Spectroscopic Information and the Data.- of X-Ray Diffraction Analysis and NMR.- 14.3 The Spectroscopic Manifestation of the Dynamic Nature of Proteins.- 14.3.1 Distribution of Microstates.- 14.3.2 Analysis of the Dynamics by Emission Spectrscopy.- 14.3.3 Conformational Dynamics and Functional Properties of Proteins.- 14.4 Subject and Method in Protein Spectroscopy.- References.