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Research has been conducted on Zn (super)2+ bound via coordination of the His and Cys residues. The role of these residues has been investigated and the results suggest that they may act as a switch for Zn (super)2+ dissociation in the distal finger motif of the HIV-1 nucleocapsid protein.

Quantitative analysis of the interactions of nucleocapsid protein NCp7 with d(ACGCC) and SL3 mutants has been performed to identify the nucleotide determinants in both NCp7-d(ACGCC) and NCp7-SL3 complexes. The use of steady-state and time-resolved fluorescence is described.

Analyzes the Zinc[sup 2+] binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein. Evidence of a critical role of cysteine 49 in Zinc[sup 2+] dissociation; Details on the spectroscopic properties of Cobalt[sup 2+]; Use of fluorescence of Trp37 to monitor the binding of Zinc[sup 2+].

The catalytic mechanisms of serine and cysteine peptidases are similar: the proton of the nucleophile (serine or cysteine) is transferred to the catalytic histidine, and the nucleophile attacks the substrate for cleavage. However, they differ in an important aspect: cysteine peptidases form a stable ion-pair intermediate in a stepwise mechanism, while serine peptidases follow a concerted mechanism. While it is known that a positive electrostatic potential at the active site of cysteine peptidases stabilizes the cysteine anion in the ion-pair state, the physical basis of the concerted mechanism of serine peptidases is poorly understood. In this work, we use continuum electrostatic analysis and quantum mechanical/molecular mechanical (QM/MM) simulations to demonstrate that a destabilization of an anionic serine by a negative electrostatic potential in combination with a compact active site geometry facilitates a concerted mechanism in serine peptidases. Moreover, we show that an anionic serine would destabilize the protein significantly compared to an anionic cysteine in cysteine peptidases, which underlines the necessity of a concerted mechanism for serine peptidases. On the basis of our calculations on an inactive serine mutant of a natural cysteine peptidase, we show that the energy barrier for the catalytic mechanism can be substantially decreased by introducing a negative electrostatic potential and by reducing the relevant distances indicating that these parameters are essential for the activity of serine peptidases. Our work demonstrates that the concerted mechanism of serine peptidases represents an evolutionary innovative way to perform catalysis without the energetically expensive need to stabilize the anionic serine. In contrast in cysteine peptidases, the anionic cysteine is energetically easily accessible and it is a very efficient nucleophile, making these peptidases mechanistically simple. However, a cysteine is highly oxygen sensitive, which is problematic in an aerobic environment. On the basis of the analysis in this work, we suggest that serine peptidases represent an oxygen-insensitive alternative to cysteine peptidases.

Phytochelatins (PCs) are nonribosomal thiol-rich oligopeptides synthetized from glutathione (GSH) in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases (PCSs). Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS-like enzyme from the cyanobacterium Nostoc sp. (NsPCS) is considered to be an evolutionary precursor enzyme of genuine PCSs because it shows sufficient sequence similarity for homology to the catalytic domain of the eukaryotic PCSs and shares the peptidase activity consisting in the deglycination of GSH. In this work, we investigate the catalytic mechanism of NsPCS by combining structural, spectroscopic, thermodynamic, and theoretical techniques. We report several crystal structures of NsPCS capturing different states of the catalyzed chemical reaction: (i) the structure of the wild-type enzyme (wt-NsPCS); (ii) the high-resolution structure of the γ-glutamyl-cysteine acyl-enzyme intermediate (acyl-NsPCS); and (iii) the structure of an inactive variant of NsPCS, with the catalytic cysteine mutated into serine (C70S-NsPCS). We characterize NsPCS as a relatively slow enzyme whose activity is sensitive to the redox state of the substrate. Namely, NsPCS is active with reduced glutathione (GSH), but is inhibited by oxidized glutathione (GSSG) because the cleavage product is not released from the enzyme. Our biophysical analysis led us to suggest that the biological function of NsPCS is being a part of a redox sensing system. In addition, we propose a mechanism how PCS-like enzymes may have evolved toward genuine PCS enzymes.

The water-soluble quencher hydrodabcyl can be activated as an N -succinimidyl ester that is readily accessible from crude hydrodabcyl and storable for a long time. With primary and secondary amines, it reacts swiftly and chemoselectively, even in the presence of other competing nucleophiles such as those typically present in natural peptides. One of the three phenolic OH groups of hydrodabcyl is amenable to selective mono-Boc protection resulting in reduced polarity, advantageous to its further use in organic synthesis. The advantages of hydrodabcyl over dabcyl in spectrometric applications are exemplified by the pH dependence of its absorbance spectra., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Dark fluorescence quenchers are nonfluorescent dyes that can modulate the fluorescence signal of an appropriate fluorophore donor in a distance-dependent manner. Dark quenchers are extensively used in many biomolecular analytical applications, such as studies with fluorogenic protease substrates or nucleic acids probes. A very popular dark fluorescence quencher is dabcyl, which is a hydrophobic azobenzene derivative. However, its insolubility in water may constitute a major drawback, especially during the investigation of biochemical systems whose natural solvent is water. We designed and synthesized a new azobenzene-based dark quencher with excellent solubility in aqueous media, which represents a superior alternative to the much-used dabcyl. The advantage of hydrodabcyl over dabcyl is exemplarily demonstrated for the cleavage of the fluorogenic substrate hydrodabcyl-Ser-Phe-EDANS by the proteases thermolysin and papain.

In this work, we calculate the protonation probabilities of titratable residues of bovine rhodopsin using the Poisson-Boltzmann equation. We also consider the influence of the membrane potential. Our results indicate that at physiological pH, the titratable groups directly involved in photosensing, namely Glu113, Glu181 and the retinal Schiff base, are charged. In contrast, the residues Asp83, Glu122 and His211, which are buried in the membrane, are uncharged. However, as these later residues are localized in the middle of the membrane, they are exposed to the membrane potential more strongly, which may have important functional implications. Despite of their large distance, Asp83 and Glu122 interact relatively strongly. As these two residues are in contact with opposite sides of the membrane, the membrane potential has different effects on them, which allows an enhancement of the membrane potential signal. An analysis of the different contributions to the protonation energy indicates that conformational changes that reduce the desolvation penalty of Asp83, Glu122 and His211 may lead to a complex protonation pattern change that allows an influence of the membrane potential on the function of rhodopsin. The high degree of evolutionary conservation of these three buried residues supports the idea of their functional importance. Our results are in-line with many experimental findings and lead to new ideas that can be experimentally tested., (© 2017 The American Society of Photobiology.)

In this article, we review a microstate model that uses protonation and redox microstates in order to understand the complex pH and redox titration of proteins and other polyelectrolytes. From this model, it becomes obvious that it is impossible to assign pK(a) values or redox potentials to individual protonatable or redox-active sites in a protein in which many of such sites interact. Instead each site is associated with many microscopic equilibrium constants that may lead to irregular or even non-monotonic titration curves of some groups. The microstate model provides a closed theoretical framework to discuss such phenomena.

The thermodynamics of Zn(2+) binding to three peptides corresponding to naturally occurring Zn-binding sequences in transcription factors have been quantified with isothermal titration calorimetry (ITC). These peptides, the third zinc finger of Sp1 (Sp1-3), the second zinc finger of myelin transcription factor 1 (MyT1-2), and the second Zn-binding sequence of the DNA-binding domain of glucocorticoid receptor (GR-2), bind Zn(2+) with Cys(2)His(2), Cys(2)HisCys, and Cys(4) coordination, respectively. Circular dichroism confirms that Sp1-3 and MyT1-2 have considerable and negligible Zn-stabilized secondary structure, respectively, and indicate only a small amount for GR-2. The pK(a)'s of the Sp1-3 cysteines and histidines were determined by NMR and used to estimate the number of protons displaced by Zn(2+) at pH 7.4. ITC was also used to determine this number, and the two methods agree. Subtraction of buffer contributions to the calorimetric data reveals that all three peptides have a similar affinity for Zn(2+), which has equal enthalpy and entropy components for Sp1-3 but is more enthalpically disfavored and entropically favored with increasing Cys ligands. The resulting enthalpy-entropy compensation originates from the Zn-Cys coordination, as subtraction of the cysteine deprotonation enthalpy results in a similar Zn(2+)-binding enthalpy for all three peptides, and the binding entropy tracks with the number of displaced protons. Metal and protein components of the binding enthalpy and entropy have been estimated. While dominated by Zn(2+) coordination to the cysteines and histidines, other residues in the sequence affect the protein contributions that modulate the stability of these motifs.

Charge transfer through biological macromolecules is essential for many biological processes such as for instance photosynthesis and respiration. In these processes, protons or electrons are transferred between titratable residues or redox-active cofactors, respectively. Often their transfer is tightly coupled. Computational methods based on continuum electrostatics are widely used in theoretical biochemistry to analyze the function of even very complex biochemical systems. These methods allow one to consider the pH and the redox potential of the solution as well as explicitly considering membrane potentials in the calculations. Combining continuum electrostatic calculations with a statistical thermodynamic analysis, it is possible to calculate equilibrium parameters such as protonation or oxidation probabilities. Moreover, it is also possible to simulate reaction kinetics by using parameters calculated from continuum electrostatics. One needs to consider that the transfer rate between two sites depends on the current charge configuration of neighboring sites. We formulate the kinetics of charge transfer systems in a microstate formalism. A unique transfer rate constant can be assigned to the interconversion of microstates. Mutual interactions between sites participating in the transfer reactions are naturally taken into account. This formalism is applied to the kinetics of electron transfer in the tetraheme-subunit and the special pair of the reaction center of Blastochloris viridis. It is shown that continuum electrostatic calculations can be used in combination with an existing rate law to obtain electron transfer rate constants. The relaxation electron transfer kinetics after photo-oxidation of the special pair of photosynthetic reaction center is simulated by a microstate formalism and it is shown to be in good agreement with experimental data. A flux analysis is used to follow the individual electron transfer steps. This method of simulating the complex kinetics of biomolecules based on structural data is a first step on the way from structural biology to systems biology.

Because of their central importance for understanding enzymatic mechanisms, pK(a) values are of great interest in biochemical research. It is common practice to determine pK(a) values of amino acid residues in proteins from NMR or FTIR titration curves by determining the pH at which the protonation probability is 50%. The pH dependence of the free energy required to protonate this residue is then determined from the linear relationship DeltaG(prot) = RT ln 10 (pH-pK(a)), where R is the gas constant and T the absolute temperature. However, this approach neglects that there can be important electrostatic interactions in the proteins that can shift the protonation energy. Even if the titration curves seem to have a standard sigmoidal shape, the protonation energy of an individual site in a protein may depend nonlinearly on pH. To account for this nonlinear dependence, we show that it is required to introduce pK(a) values for individual sites in proteins that depend on pH. Two different definitions are discussed. One definition is based on a rearranged Henderson-Hasselbalch equation, and the other definition is based on an equation that was used by Tanford and Roxby to approximate titration curves of proteins. In the limiting case of weak interactions, the two definitions lead to nearly the same pK(a) value. We discuss how these two differently defined pK(a) values are related to the free energy change required to protonate a site. Using individual site pK(a) values, we demonstrate on simple model systems that the interactions between protonatable residues in proteins can help to maintain the energy required to protonate a site in the protein nearly constant over a wide pH range. We show with the example of RNase T1 that such a mechanism to keep the protonation energy constant is used in enzymes. The pH dependence of pK(a) values may be an important concept in enzyme catalysis. Neglecting this concept, important features of enzymes may be missed, and the enzymatic mechanism may not be fully understood.

Computational methods based on continuum electrostatics are widely used in theoretical biochemistry to analyze the function of proteins. Continuum electrostatic methods in combination with quantum chemical and molecular mechanical methods can help to analyze even very complex biochemical systems. In this article, applications of these methods to proteins involved in photosynthesis are reviewed. After giving a short introduction to the basic concepts of the continuum electrostatic model based on the Poisson-Boltzmann equation, we describe the application of this approach to the docking of electron transfer proteins, to the comparison of isofunctional proteins, to the tuning of absorption spectra, to the analysis of the coupling of electron and proton transfer, to the analysis of the effect of membrane potentials on the energetics of membrane proteins, and to the kinetics of charge transfer reactions. Simulations as those reviewed in this article help to analyze molecular mechanisms on the basis of the structure of the protein, guide new experiments, and provide a better and deeper understanding of protein functions.

HIV-1 nucleocapsid protein, NCp7, contains two highly conserved CCHC zinc fingers. Binding of Zn(2+) drives NCp7 from an unfolded to a highly folded structure that is critical for its functions. Using the intrinsic fluorescence of Trp(37), we investigated, by the stopped-flow technique, the folding of NCp7 distal finger through the pH dependence of its Zn(2+) association and dissociation kinetics. Zn(2+) binding was found to involve four different paths associated with the four deprotonated states of the finger. Each binding path involves the rapid formation of an intermediate complex that is subsequently rearranged and stabilized in a rate-limiting step. The equilibrium and kinetic rate constants of the full Zn(2+)-binding process have been determined. At neutral pH, the preferential pathway for the Zn(2+)-driven folding implies Zn(2+) binding to the deprotonated Cys(36) and His(44) residues, in the bidentate state of the finger. The resulting intermediate is then converted with a rate constant of 500 s(-1) into a more suitably folded form, probably through a rearrangement of the peptide backbone around Zn(2+) to optimize the binding geometry. This form then rapidly leads to the final native complex, through deprotonation of Cys(39) and Cys(49) residues and intramolecular substitution of coordinated water molecules. Zn(2+) dissociation is also characterized by a multistep process and occurs fastest via the deprotonated Zn(2+)-bound bidentate state with a rate constant of 3 s(-1). Due to their critical role in folding, the intermediates identified for the first time in this study may constitute potential targets for HIV therapy.

Proton binding and release are elementary steps for the transfer of protons within proteins, which is a process that is crucial in biochemical catalysis and biological energy transduction. Local electric fields in proteins affect the proton binding energy compared to aqueous solution. In membrane proteins, also the membrane potential affects the local electrostatics and can thus be crucial for protein function. In this paper, we introduce a procedure to calculate the protonation probability of titratable sites of a membrane protein in the presence of a membrane potential. In the framework of continuum electrostatics, we use a modified Poisson-Boltzmann equation to include the influence of the membrane potential. Our method considers that in a transmembrane protein each titratable site is accessible for protons from only one side of the membrane depending on the hydrogen bond pattern of the protein. We show that the protonation of sites receiving their protons from different sides of the membrane is differently influenced by the membrane potential. In addition, the effect of the membrane potential is combined with the effect of the pH gradient resulting from proton pumping. Our method is applied to bacteriorhodopsin, a light-activated proton pump. We find that the proton pumping of this protein might be regulated by Asp115, a conserved residue for which no function has been identified yet. According to our calculations, the interaction of Asp115 with Asp85 leads to the protonation of the latter if the pH gradient or the membrane potential becomes too large. Since Asp85 is the primary proton acceptor in the photocycle, bacteriorhodopsin molecules in which Asp85 is protonated cannot pump protons. Furthermore, we estimate how the membrane potential affects the energetics of the individual proton-transfer reactions of the photocycle. Most reactions, except the initial proton transfer from the Schiff base to Asp85, are influenced. Our calculations give new insights into the mechanism with which bacteriorhodopsin senses the membrane potential and the pH gradient and how the proton pumping is regulated by these parameters.

This paper presents a theoretical analysis of the titration behavior of strongly interacting titratable residues in proteins. Strongly interacting titratable residues exist in many proteins such as for instance bacteriorhodopsin, cytochrome c oxidase, cytochrome bc(1), or the photosynthetic reaction center. Strong interaction between titratable groups can lead to irregular titration behavior. We analyze under which circumstances titration curves can become irregular. We demonstrate that conformational flexibility alone can not lead to irregular titration behavior. Strong interaction between titratable groups is a necessary, but not sufficient condition for irregular titration curves. In addition, the two interacting groups also need to titrate in the same pH-range. These two conditions together lead to irregular titration curves. The mutation of a single residue within a cluster of interacting titratable residues can influence the titration behavior of the other titratable residues in the cluster. We demonstrate this effect on a cluster of four interacting residues. This example underlines that mutational studies directed at identifying the role of a certain titratable residue in a cluster of interacting residues should always be accompanied by an analysis of the effect of the mutation on the titration behavior of the other residues.

In response to DNA strand breaks in the genome of higher eukaryotes, poly(ADP-ribose)polymerase 1 (PARP-1) catalyses the covalent attachment of ADP-ribose units from NAD(+) to various nuclear acceptor proteins including PARP-1 itself. This post-translational modification affecting proteins involved in chromatin architecture and in DNA repair plays a critical role in cell survival as well as in caspase-independent cell death. Although PARP-1 has been best-studied for its role in genome stability, several recent reports have demonstrated its role in the regulation of transcription. In this study, fluorescence spectroscopy and biochemical techniques are used to investigate the association of the amino-terminal DNA-binding domain of human PARP-1 (hPARP-1 DBD) with various DNA substrates, characterized by different DNA ends and sequence features (5'- or 3'-recessed end, double strands, telomeric repeats, and the palindromic sequence of a Not I restriction site). The correlation between the binding mode of hPARP-1 DBD to the DNA oligoduplexes and the enzymatic activation of hPARP-1 is analyzed. We show that hPARP-1 DBD binds a 5'-recessed DNA end cooperatively with a stoichiometry of two proteins per DNA molecule. In contrast, a 1:1 stoichiometry is found in the presence of a 3'-recessed end and double-strand DNA. A palindromic structure like the Not I restriction site is shown to induce protein dimerization and high enzymatic activation, suggesting that it can represent a recognition element for hPARP-1 in undamaged cells. Protein dimerization is found to be a requisite for high enzymatic activity. Taken together, our data allow further characterization of the features of hPARP-1 recognition in damaged cells and bring additional evidence that hPARP-1 may also play a role in undamaged cells.

The kinetics of Zn(2+) binding by two point-mutated forms of the HIV-1 NCp7 C-terminal zinc finger, each containing tridentate binding motif HCC [Ser49(35-50)NCp7] or CCC [Ala44(35-50)NCp7], has been studied by stopped-flow spectrofluorimetry. Both the formation and dissociation rate constants of the complexes between Zn(2+) and the two model peptides depend on pH. The results are interpreted on the basis of a multistep reaction model involving three Zn(2+) binding paths due to three deprotonated states of the coordinating motif, acting as monodentate, bidentate, and tridentate ligands. For Ser49(35-50)NCp7 around neutral pH, binding preferentially occurs via the deprotonated Cys36 in the bidentate state also involving His44. The binding rate constants for the monodentate and bidentate states are 1 x 10(6) and 3.9 x 10(7) M(-)(1) s(-)(1), respectively. For Ala44(35-50)NCp7, intermolecular Zn(2+) binding predominantly occurs via the deprotonated Cys36 in the monodentate state with a rate constant of 3.6 x 10(7) M(-)(1) s(-)(1). In both mutants, the final state of the Zn(2+) complex is reached by subsequent stepwise ligand deprotonation and intramolecular substitution of coordinated water molecules. The rate constants for the intermolecular binding paths of the bidentate and tridentate states of Ala44(35-50)NCp7 and of the tridentate state of Ser49(35-50)NCp7 are much smaller than expected according to electrostatic considerations. This is attributed to conformational constraints required to achieve proper metal coordination during folding. The dissociation of Zn(2+) from both peptides is again characterized by a multistep process and takes place fastest via the protonated Zn(2+)-bound bidentate and monodentate states, with rate constants of approximately 0.3 and approximately 10(3) s(-)(1), respectively, for Ser49(35-50)NCp7 and approximately 4 x 10(-)(3) and approximately 500 s(-)(1), respectively, for Ala44(35-50)NCp7.

The nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers that strongly bind Zn(2+) through coordination of one His and three Cys residues. It has been suggested that NCp7 function is conformation specific since substitution of any of the zinc coordinating residues in the zinc finger motifs leads to subsequent loss of viral infectivity. To further determine the structural requirements necessary for this specific conformation, we investigated by (1)H 2D NMR and molecular dynamics simulations the structure of the distal finger motif of NCp7 in which the zinc coordinating amino acid, His 44, was substituted by a noncoordinating Ala residue. While the fold of the N-terminal part of this mutated peptide was similar to that of the native peptide, an increased lability and significant conformational changes were observed in the vicinity of the His-to-Ala mutation. Moreover, molecular dynamics simulations suggested a mechanism by which the variant peptide can bind zinc ion even though one zinc-coordinating amino acid was lacking. Using the fluorescence of the naturally occurring Trp37 residue, the binding affinity of the variant peptide to the (TG)(3) model oligonucleotide was found to be decreased by about 2 orders of magnitude with respect with the native peptide. Modeling of the DNA:NCp7 complex using structures of the variant peptide suggests that the residues forming a hydrophobic cleft in the native protein are improperly oriented for efficient DNA binding by the variant peptide.

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation, or oxidants. The resulting formation of protein-bound poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage probably via its contribution to DNA base excision repair. In this study, we investigated the association of the amino-terminal DNA binding domain of human PARP-1 (hPARP-1 DBD) with a 5' recessed oligonucleotide mimicking a telomeric DNA end. We used the fluorescence of the Trp residues naturally occurring in the zinc finger domain of hPARP-1 DBD. Fluorescence intensity and fluorescence anisotropy measurements consistently show that the binding stoichiometry is two proteins per DNA molecule. hPARP-1 was found to bind the 5' recessed DNA end with a binding constant of approximately 10(14) M(-2) if a cooperative binding model is assumed. These results indicate that hPARP-1 DBD dimerizes during binding to the DNA target site. A footprint experiment shows that hPARP-1 DBD is asymmetrically positioned at the junction between the double-stranded and the single-stranded telomeric repeat. The largest contribution to the stability of the complex is given by nonionic interactions. Moreover, time-resolved fluorescence measurements are in line with the involvement of one Trp residue in the stacking interaction with DNA bases. Taken together, our data open new perspectives for interpretation of the selective binding of hPARP-1 to the junction between double- and single-stranded DNA.

Time-resolved fluorescence study of single tryptophan-containing proteins, nuclease, ribonuclease T1, protein G, glucagon, and mastoparan, has been carried out. Three different methods were used for the analysis of fluorescence decays: the iterative reconvolution method, as reviewed and developed in our laboratory, the maximum entropy method, and the recent method that we called "energy transfer" method. All the proteins show heterogeneous fluorescence kinetics (multiexponential decay). The origin of this heterogeneity is interpreted in terms of current theories of electron transfer process, which treat the electron transfer process as a radiationless transition. The theoretical electron transfer rate was calculated assuming the peptide bond carbonyl as the acceptor site. The good agreement between experimental and theoretical electron-transfer rates leads us to suggest that the electron-transfer process is the principal quenching mechanism of Trp fluorescence in proteins, resulting in heterogeneous fluorescence kinetics. Furthermore, the origin of apparent homogeneous fluorescence kinetics (monoexponential decay) in some proteins also can be explained on the basis of electron-transfer mechanism.

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp residues are located from positions 159 to 167, has been engineered to investigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluorescence anisotropy decay of most mutants can be described by the sum of three exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The shortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within the rotameric chi(1) x chi(2) potential wells contribute to the picosecond depolarization process, whereas the subnanosecond and nanosecond depolarization processes are related to backbone dynamics. In the absence of inducer, the order parameters of these motions, about 0.90 and 0.80 for most positions, indicate limited flexibility of the loop backbone. Anhydrotetracycline binding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the mechanism of allosteric transition.

We analysed the conformational states of free, tet operator-bound and anhydrotetracycline-bound Tet repressor employing a Trp-scanning approach. The two wild-type Trp residues in Tet repressor were replaced by Tyr or Phe and single Trp residues were introduced at each of the positions 162-173, representing part of an unstructured loop and the N-terminal six residues of alpha-helix 9. All mutants retained in vivo inducibility, but anhydrotetracycline-binding constants were decreased up to 7.5-fold when Trp was in positions 169, 170 and 173. Helical positions (168-173) differed from those in the loop (162-167) in terms of their fluorescence emission maxima, quenching rate constants with acrylamide and anisotropies in the free and tet operator-complexed proteins. Trp fluorescence emission decreased drastically upon atc binding, mainly due to energy transfer. For all proteins, either free, tet operator bound or anhydrtetracycline-bound, mean fluorescence lifetimes were determined to derive quenching rate constants. Solvent-accessible surfaces of the respective Trp side chains were calculated and compared with the quenching rate constants in the anhydrotetracycline-bound complexes. The results support a model, in which residues in the loop become more exposed, whereas residues in alpha-helix 9 become more buried upon the induction of TetR by anhydrotetracycline.

Depending on the HIV-1 isolate, MN or BH10, the nucleocapsid protein, NCp7, corresponds to a 55- or 71-amino acid length product, respectively. The MN NCp7 contains a single Trp residue at position 37 in the distal zinc finger motif, and the BH10 NCp7 contains an additional Trp, at position 61 in the C-terminal chain. The time-resolved intensity decay parameters of the zinc-saturated BH10 NCp7 were determined and compared to those of single-Trp-containing derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fractional intensities) of the individual emitting Trp residues. This suggested the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studies. In the presence of tRNAPhe, taken as a RNA model, the same conclusions hold true despite the large fluorescence decrease induced by the binding of tRNAPhe. Indeed, the fluorescence of Trp37 appears almost fully quenched, in keeping with a stacking of this residue with the bases of tRNAPhe. Despite the multiple binding sites in tRNAPhe, the large prevalence of ultrashort lifetimes, associated with the stacking of Trp37, suggests that this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp61 only stacked to a small extent with tRNAPhe. The behavior of this residue in the tRNAPhe-NCp7 complexes appeared to be rather heterogeneous, suggesting that it does not constitute a major determinant in the binding process. Finally, our data suggested that the binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucleic acid sequences was largely similar.

We have studied the time-resolved fluorescence of three engineered Tet repressor (TetR) mutants bearing a single Trp residue at positions 162, 163, and 165 in the C-terminal part of the loop joining helices 8 and 9. Detailed analysis indicates that, at 20 degrees, the fluorescence decay of each Trp can be described as the sum of three exponential components with lifetimes in the 1-, 3-, and 6-ns range. Emission wavelength and temperature dependence studies are consistent with a model in which these components are due to the existence of three classes of Trp residues non-interconverting on the nanosecond timescale. Within the framework of the rotamer model, the weak temperature dependence of the lifetimes strongly suggests that the secondary structure of the loop, at least in the 162-165 range, is not altered with temperature. The equilibrium between the rotamers is characterized by an enthalpy-entropy compensation effect which strongly suggests the involvement of background structural regions of TetR in the thermodynamics of the process. The very high deltaH degrees and TdeltaS degrees observed (up to 18 kcal/ mol) should reflect the temperature-dependent conformational change of a large part of the protein which would alter the rotamer distribution of the Trp residues. Taken together, our results are consistent with the existence of (at least) two conformations of the loop and suggest a model for loop motion.