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Steven T Whitten

Publications and source records attributed to Steven T Whitten.

7 recordsLinked to original sources
PubMed2006

Revealing the nature of the native state ensemble through cold denaturation.

Recent advances in NMR methodology have enabled the structural analysis of proteins at temperatures far below the freezing point of water, thus opening a window to the cold denaturation process. Although the phenomenon of cold denaturation has been known since the mid-1970s, the freezing point of water has prevented detailed and structurally resolved studies without application of additional significant perturbations of the protein ensemble. As a result, the cold-denatured state and the process of cold denaturation have gone largely unstudied. Here, the structural and thermodynamic basis of cold denaturation is explored with emphasis placed on the insights that are uniquely ascertained from low-temperature studies. It is shown that the noncooperative cold-induced unfolding of protein results in the population of partially folded states that cannot be accessed by other techniques. The structurally resolved view of the cold denaturation process therefore can provide direct access to the cooperative substructures within the protein molecule and provide an unprecedented structurally resolved picture of the states that comprise the native state ensemble.

Animals↗
PubMed2006

Molecular mechanisms of pH-driven conformational transitions of proteins: insights from continuum electrostatics calculations of acid unfolding.

The acid unfolding of staphylococcal nuclease (SNase) is very cooperative (Whitten and García-Moreno, Biochemistry 2000;39:14292-14304). As many as seven hydrogen ions (H+) are bound preferentially by the acid-unfolded state relative to the native (N) state in the pH range 3.2-3.9. To investigate the mechanism of acid unfolding, structure-based pKa calculations were performed with a variety of continuum electrostatic methods. The calculations reproduced successfully the H+ binding properties of the N state between pH 5 and 9, but they systematically overestimated the number of H+ bound upon acid unfolding. The calculated pKa values of all carboxylic residues in the N state were more depressed than they should be. The discrepancy between the observed and the calculated H+ uptake upon acid unfolding was not improved by using high protein dielectric constants, structures relaxed with molecular dynamics, or other empirical modifications implemented previously by others to maximize agreement between measured and calculated pKa values. This suggests an important role for conformational fluctuations of the backbone as important determinants of pKa values of carboxylic groups. Because no global or subglobal conformational changes have been observed previously for SNase under acidic conditions above the acid-unfolding region, these fluctuations must be local. The acid unfolding of SNase does not seem to involve the disruption of the N state by accruement of intramolecular repulsive interactions, nor the protonation of key ion paired carboxylic residues. It is more consistent with modest contributions from many H+ binding groups, with an important role for local conformational fluctuations in the coupling between H+ binding and the global structural transition.

Acids↗
PubMed2006

Ensemble-based signatures of energy propagation in proteins: a new view of an old phenomenon.

The ability of a protein to transmit the energetic effects of binding from one site to another constitutes the underlying basis for allosterism and signal transduction. Despite clear experimental evidence indicating the ability of proteins to transmit the effects of binding, the means by which this propagation is facilitated is not well understood. Using our previously developed ensemble-based description of the equilibrium, we investigated the physical basis of energy propagation and identified several fundamental and general aspects of energetic coupling between residues in a protein. First, partitioning of a conformational ensemble into four distinct sub-ensembles allows for explanation of the range of experimentally observed coupling behaviors (i.e., positive, neutral, and negative coupling between various regions of the protein structure). Second, the relative thermodynamic properties of these four sub-ensembles define the energetic coupling between residues as either positive, neutral, or negative. Third, analysis of the structural and thermodynamic features of the states within each sub-ensemble reveals significant variability. This third result suggests that a quantitative description of energy propagation in proteins requires an understanding of the structural and energetic features of more than just one or a few low-energy states, but also of many high-energy states. Such findings illuminate the difficulty in interpreting energy propagation in proteins in terms of a structural pathway that physically links coupled sites.

Binding Sites↗
PubMed2005

Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model.

BACKGROUND: Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel) to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. METHODS: Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC) viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. RESULTS: Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye). In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected) compared to saline (12 of 12 infected) and a control gel (5 of 7 infected). CONCLUSION: These results suggest that physiologic or microbicide-induced acid immobilization and killing of infected white blood cells may be effective in preventing sexual transmission of cell-associated HIV.

Acids↗
PubMed2005

Local conformational fluctuations can modulate the coupling between proton binding and global structural transitions in proteins.

Local conformational fluctuations in proteins can affect the coupling between ligand binding and global structural transitions. This finding was established by monitoring quantitatively how the population distribution in the ensemble of microstates of staphylococcal nuclease was affected by proton binding. Analysis of acid unfolding and proton-binding data with an ensemble-based model suggests that local fluctuations: (i) can be effective modulators of ligand-binding affinities, (ii) are important determinants of the cooperativity of ligand-driven global structural transitions, and (iii) are well represented thermodynamically as local unfolding processes. These studies illustrate how an ensemble-based description of proteins can be used to describe quantitatively the interdependence of local conformational fluctuations, ligand-binding processes, and global structural transitions. This level of understanding of the relationship between conformation, energy, and dynamics is required for a detailed mechanistic understanding of allostery, cooperativity, and other complex functional and regulatory properties of macromolecules.

Binding Sites↗
PubMed2004

Thermodynamic mechanism and consequences of the polyproline II (PII) structural bias in the denatured states of proteins.

A quantitative characterization of the structure and energy of the denatured states of proteins represents the cornerstone to a molecular-level understanding of both protein stability and fold specificity. Recent studies have revealed a significant bias in unstructured peptides toward the polyproline II (P(II)) conformation, even when no prolines are present in the sequence. This indicates that the P(II) conformation is a dominant component of the denatured states of proteins, although a quantitative description of the component enthalpy and entropy functions associated with this conformation (i.e., the thermodynamic mechanism) has thus far proven elusive. An experimental system has been designed that, when analyzed with high-precision isothermal titration calorimetry, provides direct access to the residue-specific thermodynamics of the P(II) structure formation in disordered proteins and peptides. Here, it is shown that the P(II) bias is driven by a favorable and significant enthalpy (Deltah) of -1.7 kcal mol(-1) residue(-1), which is partially offset by an unfavorable entropy (TDeltas) of -0.7 kcal mol(-1) residue(-1), relative to the ensemble of disordered conformations of the molecule. In addition to impacting dramatically the interpretation of thermal denaturation experiments, these experimental values form the framework of a quantitative energetic description of the denatured states of proteins.

Animals↗