Corrections Fig. 2B shows SCP3 cells transduced with a vector constitutively expressing red fluorescent protein (RFP, Upper) and a vector expressing green fluorescent protein (GFP) under the control of a TGF-␤ responsive promoter (Lower). An erroneous, unpaired set of images was used in the ϩTGF-␤ panels of the previously published version of this figure.'' The corrected figure and its legend appear below. This correction does not affect the conclusions of the article.

An interesting example of an allosteric protein is the chaperonin GroEL. It undergoes adenosine 5'-triphosphate-induced conformational changes that are reflected in binding of adenosine 5'-triphosphate with positive cooperativity within rings and negative cooperativity between rings. Herein, correlated mutations in chaperonins are analyzed to unravel routes of allosteric communication in GroEL and in its complex with its co-chaperonin GroES. It is shown that analysis of correlated mutations in the chaperonin family can provide information about pathways of allosteric communication within GroEL and between GroEL and GroES. The results are discussed in the context of available structural, genetic, and biochemical data concerning short- and long-range interactions in the GroE system.

Coupling and orientation between anharmonic vibrations characterized with two-dimensional infrared vibrational echo spectroscopy J. Chem. Phys. 115, 10814 (2001) Heterodyned two-dimensional infrared ͑2D IR͒ spectroscopy has been used to study the amide I vibrational dynamics of a 27-residue peptide in lipid vesicles that encompasses the transmembrane domain of the T-cell receptor CD3 . Using 1 -13 Cv 18 O isotope labeling, the amide I mode of the 49-Leucine residue was spectroscopically isolated and the homogeneous and inhomogeneous linewidths of this mode were measured by fitting the 2D IR spectrum collected with a photon echo pulse sequence. The pure dephasing and inhomogeneous linewidths are 2 and 32 cm Ϫ1 , respectively. The population relaxation time of the amide I band was measured with a transient grating, and it contributes 9 cm Ϫ1 to the linewidth. Comparison of the 49-Leucine amide I mode and the amide I band of the entire CD3 peptide reveals that the vibrational dynamics are not uniform along the length of the peptide. Possible origins for the large amount of inhomogeneity present at the 49-Leucine site are discussed.

In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528], we used 2D IR spectroscopy and 1-13 C═ 18 O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3ζ transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the α-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity.

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The kallikrein-related peptidase (KLK) family of proteases is involved in many aspects of human health and disease. One member of this family, KLK4, has been implicated in cancer development and metastasis. Understanding mechanisms of inactivation are critical to developing selective KLK4 inhibitors. We have determined the X-ray crystal structures of KLK4 in complex with both sunflower trypsin inhibitor-1 (SFTI-1) and a rationally designed SFTI-1 derivative to atomic (~1 Å) resolution, as well as with bound nickel. These structures offer a structural rationalization for the potency and selectivity of these inhibitors, and together with MD simulation and computational analysis, reveal a dynamic pathway between the metal binding exosite and the active site, providing key details of a previously proposed allosteric mode of inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that have broad implications for the enzymology of the serine protease superfamily, and may potentially be exploited for the design of therapeutic inhibitors.

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.

The tetrameric M2 protein from influenza A is one of the simplest pH-gated H+ channels known, offering the potential of structurally characterizing its gating mechanism. Since the only ionizable groups in the pore are four histidines, we investigated the stability and dynamics of all six possible protonation states of the protein by using molecular dynamics. We show that while all channel protonation states are surprisingly stable, only systems with two or more charged histidines are appreciably conductive. The structural switch, from a uniprotonated to a biprotonated channel, causes an electrostatic repulsion between the charged histidines that pushes the helices apart. This results in the formation of a continuous water file that conducts protons via a H+ wire. pKa calculations place this transition at a pH of 5.6, in remarkable agreement with the experimental value. Since the conversion from uniprotonation to biprotonation occurs during endosome acidification, this explains how M2 is activated in vivo.