Probing the role of water in protein conformation and function.

Life began in a bath of water and has never escaped it. Cellular function has forced the evolution of many mechanisms ensuring that cellular water concentration has never changed significantly. To free oneself of any conceptual distinction among all small molecules, solutes and solvents, means that experiments to probe water's specific role in molecular function can be designed like any classical chemical reaction. Such an 'osmotic stress' strategy will be described in general and for an enzyme, hexokinase. Water behaves like a reactant that competes with glucose in binding to hexokinase, and modulates its conformational change and activity. This 'osmotic stress' strategy, now applied to many very different systems, shows that water plays a significant role, energetically, in most macromolecular reactions. It can be required to fill obligatory space, it dominates nearest non-specific interactions between large surfaces, it can be a reactant modulating conformational change; all this in addition to its more commonly perceived static role as an integral part of stereospecific intramolecular structure.

The effects of gramicidin on the structure of phospholipid assemblies.

Gramicidin is an antibiotic peptide that can be incorporated into the monolayers of cell membranes. Dimerization through hydrogen bonding between gramicidin monomers in opposing leaflets of the membrane results in the formation of an iontophoretic channel. Surrounding phospholipids influence the gating properties of this channel. Conversely, gramicidin incorporation has been shown to affect the structure of spontaneously formed lipid assemblies. Using small-angle x-ray diffraction and model systems composed of phospholipids and gramicidin, the effects produced by gramicidin on lipid layers were measured. These measurements explore how peptides are able to modulate the spontaneous curvature properties of phospholipid assemblies. The reverse hexagonal, H(II), phase formed by dioleoylphosphatidylethanolamine (DOPE) monolayers decreased in lattice dimension with increasing incorporation of gramicidin. This indicated that gramicidin itself was adding negative curvature to the lipid layers. In this system, gramicidin was measured to have an apparent intrinsic radius of curvature, R0pgram, of -7.1 A. The addition of up to 4 mol% gramicidin in DOPE did not result in the monolayers becoming stiffer, as measured by the monolayer bending moduli. Dioleoylphosphatidylcholine (DOPC) alone forms the lamellar (L(alpha)) phase when hydrated, but undergoes a transition into the reverse hexagonal (H(II)) phase when mixed with gramicidin. The lattice dimension decreases systematically with increased gramicidin content. Again, this indicated that gramicidin was adding negative curvature to the lipid monolayers but the mixture behaved structurally much less consistently than DOPE/gramicidin. Only at 12 mol% gramicidin in dioleoylphosphatidylcholine could an apparent radius of intrinsic curvature of gramicidin (R0pgram) be estimated as -7.4 A. This mixture formed monolayers that were very resistant to bending, with a measured bending modulus of 115 kT.

The effect of vitamin E on the structure of membrane lipid assemblies.

The effects of vitamin E on the activity of membrane-dependent enzymes suggest that it acts indirectly by modifying some properties of the lipid host. The effects of alpha-tocopherol (alpha-T) and alpha-tocopherol hemisuccinate (alpha-THS) on phospholipid monolayer structure, curvature, and bending elasticity were examined using X-ray diffraction and the osmotic stress method. These ligands were mixed with the hexagonal phase-forming lipid, dioleoylphosphatidylethanolamine (DOPE). Increasing levels up to 50 mol% alpha-T in DOPE in excess water result in a systematic decrease in the lattice dimension. Analysis of the structural changes imposed by alpha-T shows that it contributes a spontaneous radius of curvature of -13.7 A. This unusually negative value is comparable to diacylglycerols. alpha-T does not affect the bending elasticity of these monolayers. alpha-THS in its charged form decreases membrane curvature, but in its undissociated neutral form has a qualitatively similar but reduced effect on monolayer curvature, as does alpha-T. We discuss these results in terms of the local stresses such ligands would produce in the vicinity of a membrane protein, and how one might expect proteins to respond to such stress.

The lipid-water interface: revelations by osmotic stress.

Lipids at the bilayer-water interface are highly disordered and mobile, and large areas of the bilayer undergo thermal undulations. Water penetrates significantly down to the hydrocarbon chain level. This water, and water out to about 10 A from the surface, is perturbed by the lipid surface in a way that produces a strong hydration repulsion and precludes molecular contact between bilayers. Its removal costs work, but most of this water is neither a permeable barrier nor unavailable to solvate other solutes. All hydrophilic surfaces show this "hydration force." Most lipids have an excess higher free energy when packed within a bilayer membrane since in isolation they pack into high curvature assemblies with polar groups on the concave side. Osmotic stress measurements of those curved assemblies yield a measure of monolayer elastic parameters and the excess higher free energy, which likely controls embedded proteins. Osmotic stress experiments can determine whether water is energetically significant, or not, in almost any system. The osmotic effect of solutes, independent of specific binding, is to compete with lipids and proteins for water. Solute affinity for water can modify lipid packing and protein conformation, coupling lipid and protein structure and function to osmolality at the molecular level.

Total esophageal reconstruction using a tubed parascapular free flap.

A method for total esophageal reconstruction when intestinal options are no longer available is presented. The technique described utilizes the parascapular microsurgical free flap, which is tubed and interposed between the cervical esophagus and the gastric remnant in the abdomen. The technique involves a well-recognized microsurgical flap and may be added to the armamentarium for total esophageal reconstruction.

The influence of lysolipids on the spontaneous curvature and bending elasticity of phospholipid membranes.

The effects of lysolipids on phospholipid layer curvature and bending elasticity were examined using x-ray diffraction and the osmotic stress method. Lysolipids with two different head groups, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and differing hydrocarbon chains were mixed with the hexagonal-forming lipid, dioleoylphosphatidylethanolamine (DOPE). With up to 30 mole% lysolipid in DOPE, the mixture maintains the inverted hexagonal (H(II)) phase in excess water, where increasing levels of lysolipid result in a systematic increase in the H(II) lattice dimension. Analysis of the structural changes imposed by lysolipids show that, opposite to DOPE itself, which has an spontaneous radius of curvature (R(0)) of -30 A, PC lysolipids add high positive curvature, with R(0) = +38 to +60 A, depending on chain length. LysoPEs, in contrast, add very small curvatures. When both polar group and hydrocarbon chains of the added lysolipid mismatch those of DOPE, the structural effects are qualitatively different from otherwise. Such mismatched lysolipids "reshape" the effective combination molecule into a longer and more cylindrical configuration compared to those lysolipids with either matching polar group or hydrocarbon chain.

The maximally vascularized central pedicle breast reduction: evolution of a technique.

A retrospective review of patient medical records was used to evaluate a surgical procedure for reduction mammaplasty. Information obtained during this review was then used to determine areas of possible improvement in patient selection and in surgical technique. Modifications were made, and a second series of patients was treated according to the new selection criteria, and by a modified surgical technique. The medical records of the second series of patients were reviewed. The two series of patients are compared. The initial series includes a retrospective review of data collected on 43 consecutive patients treated for bilateral macromastia using the central pedicle breast reduction technique of Hester and colleagues. The results of this initial series of patients were reviewed and presented at the Northwest Society of Plastic Surgeons Meeting; Lanai, HI, in 1995. Data obtained from that review, resulted in modifications in patient selection criteria and in operative technique by the senior author (RPR). An additional 110 consecutive patients have been treated using this modified protocol. The authors review the same data for the second series of patients and compare the results of the two series. They illustrate the use of the central pedicle technique in 153 patients. They have used the central pedicle reduction mammaplasty technique of Hester and colleagues as a model to demonstrate evolution in a surgical technique after evaluation of data from the medical record. A 50% reduction in complications and a 35% reduction in operative time were obtained.

Intracellular osmotic action.

Water often acts as a critical reactant in cellular reactions. Its role can be detected by modulating water activity with osmotic agents. We describe the principles behind this 'osmotic stress' strategy, and survey the ubiquity of water effects on molecular structures that have aqueous, solute-excluding regions. These effects are seen with single-functioning molecules such as membrane channels and solution enzymes, as well as in the molecular assembly of actin, the organization of DNA and the specificity of protein/DNA interactions.

Osmotic stress, crowding, preferential hydration, and binding: A comparison of perspectives.

There has been much confusion recently about the relative merits of different approaches, osmotic stress, preferential interaction, and crowding, to describe the indirect effect of solutes on macromolecular conformations and reactions. To strengthen all interpretations of measurements and to forestall further unnecessary conceptual or linguistic confusion, we show here how the different perspectives all can be reconciled. Our approach is through the Gibbs-Duhem relation, the universal constraint on the number of ways it is possible to change the temperature, pressure, and chemical potentials of the several components in any thermodynamically defined system. From this general Gibbs-Duhem equation, it is possible to see the equivalence of the different perspectives and even to show the precise identity of the more specialized equations that the different approaches use.

Reconstruction of irradiated postpneumonectomy empyema cavity with chain-link coupled microsurgical omental and TRAM flaps.

We present the first case of complete hemithoracic reconstruction of an irradiated postpneumonectomy recurrent empyema cavity that was unresponsive to multiple conventional treatments. The procedure described used a chain-link of two coupled free flaps consisting of an omental and TRAM flap. A single abdominal donor site and single operative position are other advantages of this technique that provides sufficient volume to obviate the need for thoracoplasty even in the largest wounds.

Lessons learnt from the management of 1500 complex facial fractures.

The lessons learnt from the management of 1500 consecutive patients with complex facial fractures have been analysed. This analysis and the major changes in treatment principles have resulted in dramatic improvement in results. The application of craniofacial surgical principles, extended exposure of the craniofacial skeleton, accurate fracture reduction with rigid internal bony fixation and primary bone graft reconstruction has revolutionised the care of these patients. The reattachment of the soft tissue to the reconstructed skeleton provides the final link in this comprehensive one stage reconstruction. Analysis of results has demonstrated the majority of complications and failures to be due to inadequate exposure and reduction of fractures with bone segments rigidly plated in their unreduced position. Inadequate bone grafting and failure to re-suspend the soft tissue and canthi results in post treatment deformity even if the bony reconstruction is adequate. Adherence to the principles of reconstruction will almost always result in a one-stage correction no matter how severe the initial injury.

Water in actin polymerization.

We have addressed the question whether water is part of the G- to F-actin polymerization reaction. Under osmotic stress, the critical concentration for G-Ca-ATP actin was reduced for six different osmolytes. These results are interpreted as showing that reducing water activity favored the polymerized state. The magnitude of the effect correlated, then saturated, with increasing MW of the osmolyte and suggested that up to 10-12 fewer water molecules were associated with actin when it polymerized. By contrast, osmotic effects were insignificant for Mg-ATP actin. The nucleotide binding site of the Mg conformation is more closed than the Ca and more closely resembles the closed actin conformation in the polymerized state. These results suggest that the water may come from the cleft of the nucleotide binding site.

Results of 268 pressure sores in 158 patients managed jointly by plastic surgery and rehabilitation medicine.

Despite improvements in surgical repair of pressure sores, recurrence rates greater than 80 percent are reported, thus indicating that this difficult problem is not yet solved. Recurrence results in additional hospitalizations and increased medical expenses. Because associated general clinical and social issues are numerous for these patients, our physical medicine and rehabilitation colleagues are active participants in their perioperative medical care. In addition, the Department of Physical Medicine and Rehabilitation also directs a complete postreconstruction rehabilitation and education program for them. The results of surgically repaired pressure sores for patients managed in this collaborative fashion have not been previously reported. Pressure sore patients at the Harborview and University of Washington Medical Centers are evaluated by plastic surgery colleagues together with the Department of Physical Medicine and Rehabilitation. Patients believed to be candidates for complete postoperative rehabilitation are offered surgical repair and constitute this study cohort. Individuals who cannot cooperate with our protocol are treated nonoperatively and are not included in this study. A retrospective analysis of all 158 patients (mean age 34.5 years) operated on for 268 grade III and IV pressure sores between October of 1977 and December of 1989 was performed. Following surgical debridement and reconstruction, patients receive their principal medical care from the Department of Physical Medicine and Rehabilitation service while the Plastic Surgery Department manages the surgical wounds. Graduated patient mobilization is initiated in accord with a mutually agreed upon standardized protocol. New or primary sores numbered 174 (65 percent), and recurrent or secondary sores numbered 94 (35 percent). Mean patient follow-up was 3.7 years. The overall pressure sore recurrence rate (recurrence at the same site) was 19 percent, and the overall patient recurrence rate (previous patient developing a new sore) was 25 percent. Recurrence rates of 22 and 15 percent were noted for primary and secondary pressure sores, respectively. On most recent examination, 131 patients (83 percent) had intact pelvic and perineal skin. These results support a collaborative approach to the management of high-grade pressure sore patients. Our protocol of mutual patient evaluation followed by surgical reconstruction and postoperative rehabilitation yields notably low recurrence rates of both primary and secondary sores. In addition, the high percentage of patients who manifest long-term maintenance of skin integrity demonstrates the excellent education in personal skin and self-care that this approach provides. Not only do patients enjoy successful and durable reconstructive results, but additional hospitalizations and health care expenses implicit to pressure sore recurrence are consequently diminished. This collaborative clinical effort remains our standard of care.

Limb-salvage in reconstruction of recalcitrant pressure sores using the inferiorly based rectus abdominis myocutaneous flap.

Pressure sore closure is frequently a reconstructive challenge. This challenge is particularly evident in cases of multiply recurrent sores. In such settings, there are often opportunities to manage the recurrent wounds either by repeated advancement of previous flaps or by design of alternative ones. However, these interventions are not always feasible, and limb amputation with total thigh flap closure must be considered. A review of operative experience with seven such complex pressure sores in seven patients is presented. Each patient had previously suffered a permanent thoracic-level spinal cord injury. Prior attempts at wound closure were unsuccessful. Despite consideration of all described locoregional flaps, no limb-sparing procedure could be designed satisfactorily. As an alternative to either hip disarticulation and total thigh flap coverage or distant free-tissue transfer, we reconstructed the debrided ulcer beds with inferiorly based rectus abdominis myocutaneous flaps. Six of the seven wounds healed primarily, whereas one required repeated debridement and the addition of a gracilis muscle flap to achieve complete closure. Postoperative follow-up has ranged from 6 to 45 months. Each patient has returned to his baseline preoperative activity level with no clinical compromise of abdominal wall function. All wounds have healed. Successful application of the inferiorly based vertical rectus abdominis myocutaneous flap for cases of both recalcitrant ischial and trochanteric pressure sores is demonstrated and its consideration is advocated if no reconstructive options short of extremity amputation and total thigh flap coverage exist for such challenging sores.

Prospective trial of catheter irrigation and muscle flaps for sternal wound infection.

BACKGROUND: Sternal wound infection is a relatively rare but potentially devastating complication of open heart operations. The most common treatments after debridement are rewiring with antibiotic irrigation and muscle flaps. Here we present the results of a prospective trial to determine the appropriate roles of closed-chest catheter irrigation and muscle flap closure for sternotomy infection and to assess the effect of internal mammary artery bypass grafting on the outcome of each treatment modality. METHODS: Between 1990 and 1994, 5,658 sternotomies were performed at the University of Washington Medical Center. Sternal dehiscence occurred in 43 patients, 25 of whom had infection (overall incidence, 0.44%). Because of the infrequency of this complication, a prospective, randomized trial was developed in which the initial approach to sternal dehiscence was rewiring and catheter irrigation. Muscle flaps were used as the primary treatment if the sternum could not be restabilized or as secondary treatment if catheter irrigation failed. Wound resolution, length of hospital stay, and complications were evaluated. RESULTS: Sterile dehiscences were successfully closed with irrigation in 17 of 18 patients; the other patient required flap closure. Of the 25 patients with infection, 19 had irrigation and 6, closure with flaps primarily. In the group of infected patients, 17 of the 19 who received irrigation also had internal mammary artery bypass grafting. Irrigation failed in 15 (88.2%) of these 17 patients, and salvage was accomplished with muscle flap closure. All 6 patients with infection who were closed primarily with muscle flaps had a successful outcome. Hospitalization averaged 10.2 days when muscle flaps were used primarily and 14.3 additional days for unsuccessful irrigation. When irrigation was successful, the hospital stay averaged 11.2 days. CONCLUSIONS: Catheter irrigation should be reserved for patients without infection or patients with infection but without internal mammary artery bypass grafts in whom dehiscence occurs less than 1 month after sternotomy. All others should have closure with muscle flaps.

Comparative study of the effects of several n-alkanes on phospholipid hexagonal phases.

The effects of a series of normal alkanes (decane, dodecane, tetradecane, hexadecane, and octadecane) on the hexagonal H(II) structures containing dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) were studied using x-ray diffraction and osmotic stress. The alkanes affect structural dimensions and the monolayer intrinsic curvature and bending modulus. The alkane effects are chain-length dependent and are attributed to their different distribution within the H(II) structure. The data suggest that short-chain alkanes are more uniformly distributed within the H(II) hydrocarbon regions and change the curvature and bending modulus of the monolayer, whereas longer-chain alkanes appear confined more to the interstitial region and do not change the curvature and bending modulus.

Lipid packing stress and polypeptide aggregation: alamethicin channel probed by proton titration of lipid charge.

Lipid membranes are not passive, neutral scaffolds to hold membrane proteins. In order to examine the influence of lipid packing energetics on ion channel expression, we study the relative probabilities of alamethicin channel formation in dioleoylphosphatidylserine (DOPS) bilayers as a function of pH. The rationale for this strategy is our earlier finding that the higher-conductance states, corresponding to larger polypeptide aggregates, are more likely to occur in the presence of lipids prone to hexagonal HII-phase formation (specifically DOPE), than in the presence of lamellar L alpha-forming lipids (DOPC). In low ionic strength NaCl solutions at neutral pH, the open channel in DOPS membranes spends most of its time in states of lower conductance and resembles alamethicin channels in DOPC; at lower pH, where the lipid polar groups are neutralized, the channel probability distribution resembles that in DOPE. X-Ray diffraction studies on DOPS show a progressive decrease in the intrinsic curvature of the constituent monolayers as well as a decreased probability of HII-phase formation when the charged lipid fraction is increased. We explore how proton titration of DOPS affects lipid packing energetics, and how these energetics couple titration to channel formation.

Reducing the period of immobilization following pressure sore surgery: a prospective, randomized trial.

The cost to care for a patient with pressure sores can be exorbitant. One part of this expense results from the prolonged postoperative immobilization. Published protocols report 3 to 8 weeks of immobilization; however, there are no studies that establish longer periods to be superior. To justify our 3-week protocol, we conducted a prospective, randomized trial of 2 versus 3 weeks of postoperative immobilization. Each patient was randomized preoperatively to either 2 or 3 weeks of postoperative immobilization. A total of 42 patients with a diagnosis of paraplegia, tetraplegia, or multiple sclerosis and a solitary pressure sore were enrolled over 5 years. The complication rates in the two groups (9 of 23, or 39 percent, for the 2-week group and 9 of 19, or 47 percent, for the 3-week group) were not statistically different (p < 0.493). However, the time to mobilization was significantly reduced in the 2-week group (16.1 +/- 6.1 versus 22.9 +/- 4.9 days, p < 0.0003), as was the time to achieve sitting (21.2 versus 28.9 days, p < 0.0026). In summary, 2 weeks of postoperative immobilization following surgery is adequate for uncomplicated solitary pressure sores.

The influence of cholesterol on phospholipid membrane curvature and bending elasticity.

The behavior of dioleoylphosphatidylethanolamine (DOPE)/cholesterol/tetradecane and dioleoylphosphatidylcholine (DOPC)/cholesterol/tetradecane were examined using x-ray diffraction and the osmotic stress method. DOPE/tetradecane, with or without cholesterol, forms inverted hexagonal (HII) phases in excess water. DOPC/tetradecane forms lamellar phases without cholesterol at lower temperatures. With tetradecane, as little as 5 mol% cholesterol in DOPC induced the formation of HII phases of very large dimension. Increasing levels of cholesterol result in a systematic decrease in the HII lattice dimension for both DOPE and DOPC in excess water. Using osmotic pressure to control hydration, we applied a recent prescription to estimate the intrinsic curvature and bending modulus of the HII monolayers. The radii of the intrinsic curvature, RPO, at a pivotal plane of constant area within the monolayer were determined to be 29.4 A for DOPE/tetradecane at 22 degrees C, decreasing to 27 A at 30 mol% cholesterol. For DOPC/tetradecane at 32 degrees C, RPO decreased from 62.5 A to 40 A as its cholesterol content increased from 30 to 50 mol%. These data yielded an estimate of the intrinsic radius of curvature for pure DOPC of 87.3 A. The bending moduli kc of DOPE/tetradecane and DOPC/tetradecane, each with 30 mol% cholesterol, are 15 and 9 kT, respectively. Tetradecane itself was shown to have little effect on the bending modulus in the cases of DOPE and cholesterol/DOPE. Surprisingly, cholesterol effected only a modest increase in the kc of these monolayers, which is much smaller than estimated from its effect on the area compressibility modulus in bilayers. We discuss possible reasons for this difference.

Probing protein hydration and conformational states in solution.

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.